Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF USING INTERLEUKIN-10
FOR TREATING DISEASES AND DISORDERS
Cross-Reference to Related Application
[0001] This application claims priority benefit of US Provisional
Application Serial No.
62/275,127, filed January 5, 2016, which application is incoroproated by
reference herein in its
entirety.
Field of the Invention
[0002] This invention relates to methods of using a PEG-IL-10 in
combination with other
agents in the treatment or prevention of a diverse array of diseases and
disorders, including
cancers and immune-related disorders.
Introduction
[0003] The cytokine interleukin-10 (IL-10) is a pleiotropic cytokine that
regulates multiple
immune responses through actions on T cells, B cells, macrophages, and antigen
presenting cells
(APC). IL-10 can suppress immune responses by inhibiting expression of IL-la,
IL-10, IL-6, IL-
8, TNFa, GM-CSF and G-CSF in activated monocytes and activated macrophages,
and it also
suppresses IFN-y production by NK cells. Although IL-10 is predominantly
expressed in
macrophages, expression has also been detected in activated T cells, B cells,
mast cells, and
monocytes. In addition to suppressing immune responses, IL-10 exhibits immuno-
stimulatory
properties, including stimulating the proliferation of IL-2 ¨ and IL-4 ¨
treated thymocytes,
enhancing the viability of B cells, and stimulating the expression of MHC
class II.
[0004] Human IL-10 is a homodimer that becomes biologically inactive upon
disruption of
the non-covalent interactions between the two monomer subunits. Data obtained
from the
published crystal structure of IL-10 indicates that the functional dimer
exhibits certain similarities
to IFN-y (Zdanov et al, (1995) Structure (Lond) 3:591-601).
[0005] As a result of its pleiotropic activity, IL-10 has been linked to
a broad range of
diseases, disorders and conditions, including inflammatory conditions, immune-
related disorders,
fibrotic disorders, metabolic disorders and cancer. Clinical and pre-clinical
evaluations with IL-10
for a number of such diseases, disorders and conditions have solidified its
therapeutic potential.
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Moreover, pegylated IL-10 has been shown to be more efficacious than non-
pegylated IL-10 in
certain therapeutic settings.
SUMMARY
[0006] The present disclosure contemplates methods of using a PEG-IL-10
(e.g., rHuPEG-
IL-10), and compositions thereof, in combination with an IL-12 agent (e.g.,
rHuIL-12), and
compositions thereof, for the treatment and/or prevention of cancer-related
diseases, disorders and
conditions, and/or the symptoms thereof The methods provide the opportunity
for additive, and
perhaps synergistic, effects in the treatment and/or prevention of the cancer-
related diseases,
disorders and conditions described herein. Moreover, such combination therapy
may allow for
reduction in the amounts and/or frequencies of administration of the PEG-IL-10
and/or the IL-12
agent in which it is combined, which can result in any adverse effects being
minimized or
obviated. The combination therapy encompasses co-administration when the PEG-
IL-10 and IL-
12 agent are administered separately (e.g., two distinct pharmaceutical
compositions) or together
(e.g., one pharmaceutical composition comprising both the PEG-IL-10 and the IL-
12 agent).
[0007] As discussed in detail herein, IL-10 is deemed to be an anti-
inflammatory and
immuno-suppressive cytokine that inhibits the secretion of IFNy, IL-12 and
TNFa. It also inhibits
antigen presentation and subsequent activation of CD4+ T cells and is thus
widely considered to
be a potent immune suppressive cytokine. In clinical studies involving various
cancer patient
populations, subcutaneous administration of PEG-IL-10 as monotherapy has
yielded beneficial
results.
[0008] In particular, recent evidence indicates that PEG-IL-10 exerts
immunostimulatory
effects in context of immunoncology (Infante, J.R., et al., ASCO Meeting
Abstracts, 2015.
33(15 suppl): p. 3017). Though an understanding of the specific mechanism of
this anti-tumor
effect is not required to practice the present disclosure, the effect has been
shown to require both
CD8+ T cells and endogenous IFNy (Mumm, J.B., et al., Cancer Cell, 2011.
20(6): p. 781-96;
Emmerich, J., et al., Cancer Res, 2012. 72(14): p. 3570-81). Specifically,
CD8+ T cell exposure
to PEG-IL-10 leads to the potentiation of IFNy, Granzyme B and Perforin
secretion. The secretion
of both IFNy and Granzyme B are dependent upon T cell receptor engagement with
cognate MHC
Pantigen complexes (Chan, I.H., et al., J Interferon Cytokine Res, 2015).
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[0009] Treatment of human cancer patients with PEG-rHuIL-10 leads to
substantial
monotherapy anti-tumor responses characterized by substantial increases in
Granzyme B+
intratumoral CD8+ T cell infiltration. Concomitant with this activated CD8+
intratumoral T cell
infiltrate is a reproducible increase in the serum cytokines IFNy, IL-18, IL-
7, IL-4, GM-CSF and
the activated T cell marker FasL. In addition, treatment with PEG-rHuIL-10
decreases serum
TGF-f3. These cytokines are the hallmarks of broad spectrum immune activation.
[0010] Human IL-10 is a homodimer, and each monomer comprises 178 amino
acids, the
first 18 of which comprise a signal peptide. Unless otherwise indicated,
reference herein to human
IL-10 refers to the mature form that lacks the signal peptide, wherein each
monomer comprises
160 amino acids (see, e.g., US Patent No. 6,217,857). As used herein, the term
"PEG-IL-10"
refers to pegylated human IL-10 and variants thereof that exhibit activity
comparable to the
activity of mature human PEG-IL-10, such as pegylated murine IL-10 and
pegylated forms of
other IL-10 orthologs.
[0011] Interleukin-12 (IL-12) is a pleiotropic cytokine naturally
produced by macrophages,
B-lymphoblastoid cells, dendritic cells, and neutrophils in response to
antigenic stimulation. It is
involved in the differentiation of naïve T cells into Thl cells, can stimulate
the growth and
function of T cells, and mediates enhancement of the cytotoxic activity of NK
cells and CD8+
cytotoxic T lymphocytes. As discussed further herein, IL-12 also stimulates
the production of
IFNy and TNFa from T cells and NK cells, and reduces IL-4 mediated suppression
of IFNy. IFNy
has been shown to coordinate natural mechanisms of anticancer defense
(Jakobisiak, M. et al.
(2013) Immunol Lett 90:103-22). Due, in part, to its potent stimulation of
IFNy production, IL-12
was initially thought to represent an ideal candidate for tumor immunotherapy.
However,
systemic administration of IL-12 during initial clinical studies yielded a
very narrow therapeutic
index and resulted in an unacceptable adverse effect profile. As a result,
systemic administration
of IL-12 as a monotherapy in the oncology setting was largely deemed unviable.
(See, e.g., Teng,
M. et al. (2015) Nature Medicine 21(7):719-29).
[0012] As used herein, the terms "IL-12", "IL-12 polypeptide(s)," "IL-12-
agent(s)", "IL-
12 molecule(s)" and the like are intended to be construed broadly and include,
for example, human
and non-human IL-12 ¨ related polypeptides, including homologs, variants
(including muteins),
and fragments thereof, as well as IL-12 polypeptides having, for example, a
leader sequence (e.g.,
a signal peptide).
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[0013] In particular embodiments, the present disclosure contemplates
methods of treating
or preventing a cancer-related disease, disorder or condition in a subject,
comprising administering
to the subject: a) a therapeutically effective amount of an IL-12 agent, and
b) a therapeutically
effective amount of a PEG-IL-10; wherein the amount of the PEG-IL-10 is
sufficient to reduce the
IL-12 ¨ associated toxicity to a level less than that observed with IL-12
monotherapy.
[0014] IL-12 ¨ associated toxicities include flu-like symptoms (e.g.,
headache, fever,
chills, fatigue and arthromyalgia); hematologic toxicity, including
neutropenia and
thrombocytopenia; and hepatic toxicity, manifested by dose-dependent increases
in transaminases,
hyperbilirubinemia, and hypoalbuminemia. Other IL-12 associated adverse
effects include
inflammation of mucus membranes (e.g., oral mucositis, stomatitis and
colitis), hypotension, renal
impairment and gastrointestinal bleeding. These toxic effects have been
associated with the
secondary production of IFNy and TNFa, as well as other cytokines (e.g., IP-10
and MIG). (See,
e.g., Lasek, et al. (2014) Cancer Immunol Immunother 63:419-35; Xu, et al.
Clinical and
Developmental Immunology, volume 2010, Article ID 832454, 9 pp.); Cebon, J.,
et al., Cancer
Immun, 2003. 3: p. 7).
[0015] In other embodiments, the present disclosure contemplates methods
of treating or
preventing a cancer-related disease, disorder or condition in a subject,
comprising administering to
the subject: a) a therapeutically effective amount of an IL-12 agent; and b) a
therapeutically
effective amount of a PEG-IL-10, wherein the amount of the PEG-IL-10 is
sufficient to i) achieve
a mean IL-10 serum trough concentration of at least 1.0 ng/mL, and ii) reduce
the IL-12 ¨
associated toxicity to a level less than that observed with IL-12 monotherapy.
[0016] In still further embodiments, the present disclosure contemplates
methods of
treating or preventing a cancer-related disease, disorder or condition in a
subject, comprising
administering to the subject: a) a therapeutically effective amount of an IL-
12 agent; and b) a
therapeutically effective amount of a PEG-IL-10, wherein the amount is
sufficient to i) maintain a
mean IL-10 serum trough concentration over a period of time, wherein the mean
IL-10 serum
trough concentration is at least 1.0 ng/mL, and wherein the mean IL-10 serum
trough
concentration is maintained for at least 90% of the period of time; and ii)
reduce the IL-12 ¨
associated toxicity to a level less than that observed with IL-12 monotherapy.
[0017] The desired IL-10 serum trough concentration may depend on a
number of factors,
including the nature of the disease, disorder or condition (e.g., localized
tumor or metastatic
disease), the extent to which the subject is suffering from the malady (e.g.,
early versus late stage
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disease), whether combination therapy is being administered, and patient-
specific parameters (e.g.,
hepatic and renal function). By way of example, co-administration of PEG-IL-10
and a
chemotherapeutic agent may only require a serum trough in the -1-2 ng/mL range
in order to
observe clinical benefit, while metastatic cancer may require 6-10 ng/mL or
more to achieve
comparable clinical benefit (see, e.g., WO 2014/172392). As the skilled
artisan will appreciate,
the desired serum trough levels are context-dependent (e.g., characteristics
of a specific cancer)
and patient-specific
[0018] Thus, in particular embodiments of the present disclosure, the
mean IL-10 serum
trough concentration is at least 1.0 ng/mL, at least 1.5 ng/mL, at least 2.0
ng/mL, at least 2.5
ng/mL, at least 3.0 ng/mL, at least 3.5 ng/mL, at least 4.0 ng/mL, at least
4.5 ng/mL, at least 5.0
ng/mL, and least 5.5 ng/mL, at least 6.0 ng/mL, at least 6.5 ng/M1, at least
7.0 ng/mL, at least 7.5
ng/mL, at least 8.0 ng/mL, and least 9.0 ng/mL, at least 10.0 ng/mL, at least
11.0 ng/mL, at least
12.0 ng/mL, at least 13.0 ng/mL, at least 14.0 ng/mL, at least 15.0 ng/mL, at
least 16.0 ng/mL, at
least 17.0 ng/mL, at least 18.0 ng/mL, at least 19.0 ng/mL, at least 20.0
ng/mL, at least 21.0
ng/mL, at least 22.0 ng/mL, or greater than 22.0 ng/mL.
[0019] In further embodiments, the period of time is at least 12 hours,
at least 24 hours, at
least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least
3 weeks, at least 1
month, at least 6 weeks, at least 2 months, at least 3 months, or greater than
3 months.
[0020] In particular embodiments of the present disclosure, the mean IL-
10 serum trough
concentration is maintained for at least 85% of the period of time, at least
90%, at least 92.5%, at
least 95%, at least 98%, at least 99% or 100% of the period of time.
[0021] It is envisaged that a dosing regimen sufficient to maintain a
particular steady state
serum trough concentration (e.g., 2.0 ng/mL) may result in an initial serum
trough concentration
that is higher than the desired steady state serum trough concentration.
Because of the
pharmacodynamic and pharmacokinetic characteristics of IL-10 in a mammalian
subject, an initial
trough concentration (achieved, for example, through the administration of one
or more loading
doses followed by a series of maintenance doses) gradually but continually
decreases over a period
of time even when the dosing parameters (amount and frequency) are kept
constant. After that
period of time, the gradual but continual decrease ends and a steady state
serum trough
concentration is maintained.
[0022] By way of example, parenteral administration (e.g., SC and IV) of -
0.1 mg/kg/day
of an IL-10 agent (e.g., mIL-10) to a mouse (e.g., a C57BL/6 mouse) is
required to maintain a
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steady state serum trough concentration of, for example, 2.0 ng/mL. However,
that steady state
serum trough concentration may not be achieved until approximately 30 days
after initiation of
dosing at 0.1 mg/kg/day (and also after any loading dose(s)). Rather, after an
initial serum trough
concentration has been achieved (e.g., 2.5 ng/mL), that concentration
gradually but continually
decreases over the course of, for example, the approximately 30-day period,
after which time the
desired steady state serum trough concentration (e.g., 2.0 ng/mL) is
maintained. One of skill in
the art will be able to determine the dose needed to maintain the desired
steady state trough
concentration using, for example, ADME and patient-specific parameters.
[0023] A PEG-IL-10 of the present disclosure may comprise at least one
PEG molecule
covalently attached to at least one amino acid residue of at least one subunit
of IL-10 or comprise
a mixture (e.g., 1:1) of mono-pegylated and di-pegylated IL-10 in other
embodiments. The PEG
component of a PEG-IL-10 may have a molecular mass greater than about 5kDa,
greater than
about 10kDa, greater than about 15kDa, greater than about 20kDa, greater than
about 30kDa,
greater than about 40kDa, or greater than about 50kDa. In some embodiments,
the molecular
mass is from about 5kDa to about 10kDa, from about 5kDa to about 15kDa, from
about 5kDa to
about 20kDa, from about 10kDa to about 15kDa, from about 10kDa to about 20kDa,
from about
10kDa to about 25kDa or from about 10kDa to about 30kDa.
[0024] As indicated herein, the PEG-IL-10 is mature human PEG-IL-10 in
some
embodiments, while in other embodiments it is a variant of mature human PEG-IL-
10 that exhibits
activity comparable to the activity of mature human PEG-IL-10.
[0025] The present disclosure contemplates embodiments wherein the amount
of the PEG-
IL-10 component of the combination therapy that is administered to the subject
to treat or prevent
a cancer-related disease, disorder or condition is from 10.0 tg/kg/day to 20.0
pg/kg/day. In some
embodiments, the amount of the PEG-IL-10 administered is from 12.0 pg/kg/day
to 18.0
pg/kg/day. In some embodiments, the amount is less than 10.0 pg/kg/day, while
in other
embodiments it is greater than 20.0 tg/kg/day.
[0026] According to the present disclosure, a PEG-IL-10 may be
administered in
combination with an IL-12 agent for the treatment of a cancer-related disease,
disorder or
condition in the subject. A detailed description of the foregoing diseases,
disorders and conditions
is set forth elsewhere herein. In some embodiments, the cancer is a solid
tumor, such as a tumor
associated with breast cancer, prostate cancer, lung cancer, liver cancer,
pancreatic cancer, brain
cancer, stomach cancer, ovarian cancer, kidney cancer, testicular cancer, and
melanoma. In
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particular embodiments, the cancer is a hematological disorder, including
lymphomas such as a B-
cell lymphoma, or a leukemia.
[0027] In particular embodiments of the present disclosure, the cancer-
related disease,
disorder or condition is an immune-insensitive tumor. Tumors that are
insensitive to therapeutic
immune manipulation may be described as exhibiting the following two
characteristics: 1) active
suppression of the immune system, and 2) an inflammatory response accompanied
by the
concomitant activation of immune-suppressive mechanisms resulting from
treatment thereof
(Galon et al. (July 25 2013) Immunity 39:11-26 (PubMed PMID: 23890060)).
Examples of
immune-insensitive tumors include, but are not limited to, colon,
gastroesophageal, pancreatic and
breast cancer.
[0028] In certain embodiments of the present disclosure, the therapeutic
effects of the
PEG-IL-10 and the IL-12 agent are additive, while in other embodiments they
are synergistic.
[0029] A PEG-IL-10 and an IL-12 agent may be administered by any
effective route. In
some embodiments, they are administered by parenteral injection, including
subcutaneous
injection. In particular embodiments, a PEG-IL-10 is administered separately
from the IL-12
agent, and in other embodiments a PEG-IL-10 and an IL-12 agent are
administered together. As
indicated herein, for purposes of the present disclosure PEG-IL-10 and an IL-
12 agent are deemed
to be co-administered when administered separately or together, or in one or
more means of
delivery (e.g., a vial, IV bag or syringe).
[0030] As noted above, the various types of IL-12 agents for use in the
combination
therapies of the present disclosure include human and non-human IL-12 ¨
related polypeptides,
including homologs, variants (including muteins), and fragments thereof Also
contemplated
herein are functionally active components of the IL-12 complex, as well as the
active heterodimer
(p70). In some embodiments, the IL-12 peptides have at least 85%, at least
87%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least
98%, or at least 99% of the 306 amino acid residue human IL-12A polypeptide
and/or of the 197
amino acid residue human IL-12B polypeptide. In other embodiments, the IL-12
peptides have at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to the 306 amino
acid residue human IL-12A polypeptide and/or of the 197 amino acid residue
human IL-12B
polypeptide.
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[0031] As indicated herein, the IL-12 agent is mature human IL-12 in some
embodiments,
while in other embodiments the IL-12 agent is a variant of mature human IL-12
that exhibits
activity comparable to the activity of mature human IL-12.
[0032] Also provided herein are embodiments wherein the amount of the IL-
12 agent of
the combination therapy that is administered to the subject to treat or
prevent a cancer-related
disease, disorder or condition is from 0.01 tg/kg/day to 10.0 pg/kg/day, from
0.1 tg/kg/day to
10.0 tg/kg/day, or from 1.0 pg/kg/day to 10.0 tg/kg/day. In some embodiments,
the amount is
less than 0.01 tg/kg/day, while in other embodiments it is greater than 10.0
tg/kg/day.
[0033] In particular embodiments, the present disclosure contemplates
dosing an IL-12
agent such that the serum concentration achieves a peak and is then cleared
such that it is
essentially unmeasurable before it is administered again. In some embodiments,
IL-12 treatment
is initiated with a loading dose, followed by a series of maintenance doses,
which may be at
defined intervals. In order to avoid potential toxicities, dosing should be
adjusted such that the IL-
12 level does not exceed its maximally tolerated level. As with administration
of a PEG-IL-10,
the dose of an IL-12 agent may depend on a number of factors, including the
nature of the disease,
disorder or condition (e.g., localized tumor or metastatic disease), the
extent to which the subject
is suffering from the malady (e.g., early versus late stage disease), whether
combination therapy is
being administered, and patient-specific parameters (e.g., hepatic and renal
function).
[0034] The present disclosure includes pharmaceutical compositions
comprising a PEG-
IL-10 and an IL-12 agent as described herein, and a pharmaceutically
acceptable diluent, carrier or
excipient. In some embodiments, the PEG-IL-10 and the IL-12 agent are present
in separate
pharmaceutical compositions, each comprising a pharmaceutically acceptable
diluent, carrier or
excipient. In some embodiments, the excipient is an isotonic injection
solution. The
pharmaceutical compositions may be suitable for administration to a subject
(e.g., a human), and
may comprise one or more additional prophylactic or therapeutic agents. In
certain embodiments,
the pharmaceutical compositions are contained in one or more sterile
containers (e.g., a single- or
multi-use vial or a syringe). A kit may contain the sterile container(s), and
the kit may also
contain one or more additional sterile containers comprising at least one
additional prophylactic or
therapeutic agent or any other agent that may be used in pharmacological
therapy. One or more
additional prophylactic or therapeutic agents may be administered prior to,
simultaneously with, or
subsequent to the PEG-IL-10 and IL-12 agent.
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[0035] Additional prophylactic or therapeutic agents (also referred to
herein as
supplementary agents and the like) that may be used with the methods of
treating and/or
preventing a cancer-related disease, disorder or condition include any agent
that may provide some
therapeutic benefit. By way of example, but not limitation, a prophylactic or
therapeutic agent
may be a chemotherapeutic agent, an immune- or inflammation-related agent, a
metabolic agent,
an antiviral agent or an anti-thrombotic agent. The methods of the present
disclosure may also be
used in combination with non-pharmacological agents (e.g., radiology).
[0036] In particular embodiments, the additional prophylactic or
therapeutic agent is a
chemotherapeutic agent, examples of which are set forth herein. In some
embodiments, the
chemotherapeutic agent is a platinum-based antineoplastic, also referred to as
a platinum
coordination complex. These platinum-based antineoplastic agents crosslink
DNA, thereby
inhibiting DNA repair and/or DNA synthesis in cancer cells. Examples of such
agents include
cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin and
triplatin.
[0037] Methods and models for optimizing dosing regimens for the PEG-IL-
10 and IL-12
agents described herein are also contemplated by embodiments of the present
disclosure. In other
embodiments, the present disclosure contemplates methods for the
identification of specific patient
populations that are optimally suited for the combination therapies described
herein. In some
embodiments, the existence and/or extent of certain biomarkers can find
utility in such methods.
[0038] Other aspects and embodiments will be apparent to the skilled
artisan after
reviewing the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 depicts the amino acid sequences of Human IL-12, Chain A
(SEQ ID
NO:1); and Human IL-12, Chain B (SEQ ID NO:2).
[0040] FIG. 2 depicts the effect of PEG-rMuIL-10 (1 mg/kg) and/or rMuIL-
12 (0.05, 0.1,
or 0.5 mg/kg) administered SC daily for 21 days as monotherapy or as
combination therapy in 4T1
tumor-bearing mice. Tumor weights were assessed after study completion.
[0041] FIGS. 3A and 3B depict the effect of PEG-rMuIL-10 (1 mg/kg) and/or
rMuIL-12
(0.05, 0.1, or 0.5 mg/kg) administered SC daily as monotherapy or as
combination therapy to 4T1
tumor-bearing mice on serum IFNy (FIG. 3A) and on serum TNFa (FIG. 3B). Serum
IFNy and
TNFa levels were assessed after 9 days of dosing, 4 hrs after dose
administration.
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DETAILED DESCRIPTION
[0042] Before the present disclosure is further described, it is to be
understood that the
disclosure is not limited to the particular embodiments set forth herein, and
it is also to be
understood that the terminology used herein is for the purpose of describing
particular
embodiments only, and is not intended to be limiting.
[0043] Where a range of values is provided, it is understood that each
intervening value, to
the tenth of the unit of the lower limit unless the context clearly dictates
otherwise, between the
upper and lower limit of that range and any other stated or intervening value
in that stated range, is
encompassed within the invention. The upper and lower limits of these smaller
ranges can
independently be included in the smaller ranges, and are also encompassed
within the invention,
subject to any specifically excluded limit in the stated range. Where the
stated range includes one
or both of the limits, ranges excluding either or both of those included
limits are also included in
the invention. Unless defined otherwise, all technical and scientific terms
used herein have the
same meaning as commonly understood by one of ordinary skill in the art to
which this invention
belongs.
[0044] It must be noted that as used herein and in the appended claims,
the singular forms
"a," "an," and "the" include plural referents unless the context clearly
dictates otherwise. It is
further noted that the claims may be drafted to exclude any optional element.
As such, this
statement is intended to serve as antecedent basis for use of such exclusive
terminology such as
"solely," "only" and the like in connection with the recitation of claim
elements, or use of a
"negative" limitation.
[0045] The publications discussed herein are provided solely for their
disclosure prior to
the filing date of the present application. Further, the dates of publication
provided may be
different from the actual publication dates, which may need to be
independently confirmed.
Overview
[0046] As described herein, the inventors of the present disclosure have
discovered that
co-administration of PEG-IL-10 and IL-12 under certain conditions and
parameters can temper IL-
12's untoward adverse effects while still retaining its potent anti-tumor
activity. In view of that
finding, the present disclosure contemplates methods of using a PEG-IL-10
(e.g., rHuPEG-IL-10),
and compositions thereof, in combination with an IL-12 agent (e.g., rHuIL-12),
and compositions
thereof, for the treatment and/or prevention of cancer-related diseases,
disorders and conditions,
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and/or the symptoms thereof. The methods comprise particular dosing regimens
and provide the
opportunity for additive or synergistic effects in the treatment and/or
prevention of the disorders
described herein.
[0047] It should be noted that any reference to "human" in connection
with the
polypeptides and nucleic acid molecules of the present disclosure is not meant
to be limiting with
respect to the manner in which the polypeptide or nucleic acid is obtained or
the source, but rather
is only with reference to the sequence as it can correspond to a sequence of a
naturally occurring
human polypeptide or nucleic acid molecule. In addition to the human
polypeptides and the
nucleic acid molecules which encode them, the present disclosure contemplates
IL-10 ¨ and IL-12
¨ related polypeptides and corresponding nucleic acid molecules from other
species.
Definitions
[0048] Unless otherwise indicated, the following terms are intended to
have the meaning
set forth below. Other terms are defined elsewhere throughout the
specification.
[0049] The terms "patient" or "subject" are used interchangeably to refer
to a human or a
non-human animal (e.g., a mammal).
[0050] The terms "administration", "administer" and the like, as they
apply to, for
example, a subject, cell, tissue, organ, or biological fluid, refer to contact
of, for example, IL-10 or
PEG-IL-10), a nucleic acid (e.g., a nucleic acid encoding native human IL-10);
a pharmaceutical
composition comprising the foregoing, or a diagnostic agent to the subject,
cell, tissue, organ, or
biological fluid. In the context of a cell, administration includes contact
(e.g., in vitro or ex vivo)
of a reagent to the cell, as well as contact of a reagent to a fluid, where
the fluid is in contact with
the cell.
[0051] The terms "treat", "treating", treatment" and the like refer to a
course of action
(such as administering IL-10 or a pharmaceutical composition comprising IL-10)
initiated after a
disease, disorder or condition, or a symptom thereof, has been diagnosed,
observed, and the like so
as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily
or permanently, at
least one of the underlying causes of a disease, disorder, or condition
afflicting a subject, or at
least one of the symptoms associated with a disease, disorder, condition
afflicting a subject. Thus,
treatment includes inhibiting (e.g., arresting the development or further
development of the
disease, disorder or condition or clinical symptoms association therewith) an
active disease. The
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terms may also be used in other contexts, such as situations where IL-10 or
PEG-IL-10 contacts an
IL-10 receptor in, for example, the fluid phase or colloidal phase.
[0052] The term "in need of treatment" as used herein refers to a
judgment made by a
physician or other caregiver that a subject requires or will benefit from
treatment. This judgment
is made based on a variety of factors that are in the realm of the physician's
or caregiver's
expertise.
[0053] The terms "prevent", "preventing", "prevention" and the like refer
to a course of
action (such as administering IL-10 or a pharmaceutical composition comprising
IL-10) initiated
in a manner (e.g., prior to the onset of a disease, disorder, condition or
symptom thereof) so as to
prevent, suppress, inhibit or reduce, either temporarily or permanently, a
subject's risk of
developing a disease, disorder, condition or the like (as determined by, for
example, the absence of
clinical symptoms) or delaying the onset thereof, generally in the context of
a subject predisposed
to having a particular disease, disorder or condition. In certain instances,
the terms also refer to
slowing the progression of the disease, disorder or condition or inhibiting
progression thereof to a
harmful or otherwise undesired state.
[0054] The term "in need of prevention" as used herein refers to a
judgment made by a
physician or other caregiver that a subject requires or will benefit from
preventative care. This
judgment is made based on a variety of factors that are in the realm of a
physician's or caregiver's
expertise.
[0055] The phrase "therapeutically effective amount" refers to the
administration of an
agent to a subject, either alone or as part of a pharmaceutical composition
and either in a single
dose or as part of a series of doses, in an amount capable of having any
detectable, positive effect
on any symptom, aspect, or characteristic of a disease, disorder or condition
when administered to
the subject. The therapeutically effective amount can be ascertained by
measuring relevant
physiological effects, and it can be adjusted in connection with the dosing
regimen and diagnostic
analysis of the subject's condition, and the like. By way of example,
measurement of the amount
of inflammatory cytokines produced following administration can be indicative
of whether a
therapeutically effective amount has been used.
[0056] The phrase "in a sufficient amount to effect a change" means that
there is a
detectable difference between a level of an indicator measured before (e.g., a
baseline level) and
after administration of a particular therapy. Indicators include any objective
parameter (e.g.,
serum concentration of IL-10) or subjective parameter (e.g., a subject's
feeling of well-being).
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[0057] The term "small molecules" refers to chemical compounds having a
molecular
weight that is less than about 10kDa, less than about 2kDa, or less than about
lkDa. Small
molecules include, but are not limited to, inorganic molecules, organic
molecules, organic
molecules containing an inorganic component, molecules comprising a
radioactive atom, and
synthetic molecules. Therapeutically, a small molecule can be more permeable
to cells, less
susceptible to degradation, and less likely to elicit an immune response than
large molecules.
[0058] The term "ligand" refers to, for example, peptide, polypeptide,
membrane-
associated or membrane-bound molecule, or complex thereof, that can act as an
agonist or
antagonist of a receptor. "Ligand" encompasses natural and synthetic ligands,
e.g., cytokines,
cytokine variants, analogs, muteins, and binding compositions derived from
antibodies. "Ligand"
also encompasses small molecules, e.g., peptide mimetics of cytokines and
peptide mimetics of
antibodies. The term also encompasses an agent that is neither an agonist nor
antagonist, but that
can bind to a receptor without significantly influencing its biological
properties, e.g., signaling or
adhesion. Moreover, the term includes a membrane-bound ligand that has been
changed, e.g., by
chemical or recombinant methods, to a soluble version of the membrane-bound
ligand. A ligand
or receptor can be entirely intracellular, that is, it can reside in the
cytosol, nucleus, or some other
intracellular compartment. The complex of a ligand and receptor is termed a
"ligand-receptor
complex."
[0059] The terms "inhibitors" and "antagonists", or "activators" and
"agonists", refer to
inhibitory or activating molecules, respectively, for example, for the
activation of, e.g., a ligand,
receptor, cofactor, gene, cell, tissue, or organ. Inhibitors are molecules
that decrease, block,
prevent, delay activation, inactivate, desensitize, or down-regulate, e.g., a
gene, protein, ligand,
receptor, or cell. Activators are molecules that increase, activate,
facilitate, enhance activation,
sensitize, or up-regulate, e.g., a gene, protein, ligand, receptor, or cell.
An inhibitor can also be
defined as a molecule that reduces, blocks, or inactivates a constitutive
activity. An "agonist" is a
molecule that interacts with a target to cause or promote an increase in the
activation of the target.
An "antagonist" is a molecule that opposes the action(s) of an agonist. An
antagonist prevents,
reduces, inhibits, or neutralizes the activity of an agonist, and an
antagonist can also prevent,
inhibit, or reduce constitutive activity of a target, e.g., a target receptor,
even where there is no
identified agonist.
[0060] The terms "modulate", "modulation" and the like refer to the
ability of a molecule
(e.g., an activator or an inhibitor) to increase or decrease the function or
activity of a PEG-IL-10
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(or the nucleic acid molecules encoding them), either directly or indirectly;
or to enhance the
ability of a molecule to produce an effect comparable to that of a PEG-IL-10.
The term
"modulator" is meant to refer broadly to molecules that can effect the
activities described above.
By way of example, a modulator of, e.g., a gene, a receptor, a ligand, or a
cell, is a molecule that
alters an activity of the gene, receptor, ligand, or cell, where activity can
be activated, inhibited, or
altered in its regulatory properties. A modulator can act alone, or it can use
a cofactor, e.g., a
protein, metal ion, or small molecule. The term "modulator" includes agents
that operate through
the same mechanism of action as IL-10 (i.e., agents that modulate the same
signaling pathway as
IL-10 in a manner analogous thereto) and are capable of eliciting a biological
response comparable
to (or greater than) that of IL-10.
[0061] Examples of modulators include small molecule compounds and other
bioorganic
molecules. Numerous libraries of small molecule compounds (e.g., combinatorial
libraries) are
commercially available and can serve as a starting point for identifying a
modulator. The skilled
artisan is able to develop one or more assays (e.g., biochemical or cell-based
assays) in which such
compound libraries can be screened in order to identify one or more compounds
having the
desired properties; thereafter, the skilled medicinal chemist is able to
optimize such one or more
compounds by, for example, synthesizing and evaluating analogs and derivatives
thereof.
Synthetic and/or molecular modeling studies can also be utilized in the
identification of an
Activator.
[0062] The "activity" of a molecule can describe or refer to the binding
of the molecule to
a ligand or to a receptor; to catalytic activity; to the ability to stimulate
gene expression or cell
signaling, differentiation, or maturation; to antigenic activity; to the
modulation of activities of
other molecules; and the like. The term can also refer to activity in
modulating or maintaining
cell-to-cell interactions (e.g., adhesion), or activity in maintaining a
structure of a cell (e.g., a cell
membrane). "Activity" can also mean specific activity, e.g., [catalytic
activity]/[mg protein], or
[immunological activity]/[mg protein], concentration in a biological
compartment, or the like. The
term "proliferative activity" encompasses an activity that promotes, that is
necessary for, or that is
specifically associated with, for example, normal cell division, as well as
cancer, tumors,
dysplasia, cell transformation, metastasis, and angiogenesis.
[0063] As used herein, "comparable", "comparable activity", "activity
comparable to",
"comparable effect", "effect comparable to", and the like are relative terms
that can be viewed
quantitatively and/or qualitatively. The meaning of the terms is frequently
dependent on the
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context in which they are used. By way of example, two agents that both
activate a receptor can
be viewed as having a comparable effect from a qualitative perspective, but
the two agents can be
viewed as lacking a comparable effect from a quantitative perspective if one
agent is only able to
achieve 20% of the activity of the other agent as determined in an art-
accepted assay (e.g., a dose-
response assay) or in an art-accepted animal model. When comparing one result
to another result
(e.g., one result to a reference standard), "comparable" frequently means that
one result deviates
from a reference standard by less than 35%, by less than 30%, by less than
25%, by less than 20%,
by less than 15%, by less than 10%, by less than 7%, by less than 5%, by less
than 4%, by less
than 3%, by less than 2%, or by less than 1%. In particular embodiments, one
result is comparable
to a reference standard if it deviates by less than 15%, by less than 10%, or
by less than 5% from
the reference standard. By way of example, but not limitation, the activity or
effect can refer to
efficacy, stability, solubility, or immunogenicity.
[0064] The term "response," for example, of a cell, tissue, organ, or
organism,
encompasses a change in biochemical or physiological behavior, e.g.,
concentration, density,
adhesion, or migration within a biological compartment, rate of gene
expression, or state of
differentiation, where the change is correlated with activation, stimulation,
or treatment, or with
internal mechanisms such as genetic programming. In certain contexts, the
terms "activation",
"stimulation", and the like refer to cell activation as regulated by internal
mechanisms, as well as
by external or environmental factors; whereas the terms "inhibition", "down-
regulation" and the
like refer to the opposite effects.
[0065] The terms "polypeptide," "peptide," and "protein", used
interchangeably herein,
refer to a polymeric form of amino acids of any length, which can include
genetically coded and
non-genetically coded amino acids, chemically or biochemically modified or
derivatized amino
acids, and polypeptides having modified polypeptide backbones. The terms
include fusion
proteins, including, but not limited to, fusion proteins with a heterologous
amino acid sequence;
fusion proteins with heterologous and homologous leader sequences; fusion
proteins with or
without N-terminus methionine residues; fusion proteins with immunologically
tagged proteins;
and the like.
[0066] It will be appreciated that throughout this disclosure reference
is made to amino
acids according to the single letter or three letter codes. For the reader's
convenience, the single
and three letter amino acid codes are provided below:
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G Glycine Gly P Proline Pro
A Alanine Ala V Valine Val
L Leucine Leu I Isoleucine Ile
M Methionine Met C Cysteine Cys
F Phenylalanine Phe Y Tyrosine Tyr
W Tryptophan Trp H Histidine His
K Lysine Lys R Arginine Arg
Q Glutamine Gln N Asparagine Asn
E Glutamic Acid Glu D
Aspartic Acid Asp
S Serine Ser T Threonine Thr
[0067] As used herein, the term "variant" encompasses naturally-occurring
variants and
non-naturally-occurring variants. Naturally-occurring variants include
homologs (polypeptides
and nucleic acids that differ in amino acid or nucleotide sequence,
respectively, from one species
to another), and allelic variants (polypeptides and nucleic acids that differ
in amino acid or
nucleotide sequence, respectively, from one individual to another within a
species). Non-
naturally-occurring variants include polypeptides and nucleic acids that
comprise a change in
amino acid or nucleotide sequence, respectively, where the change in sequence
is artificially
introduced (e.g., muteins); for example, the change is generated in the
laboratory by human
intervention ("hand of man"). Thus, herein a "mutein" refers broadly to
mutated recombinant
proteins that usually carry single or multiple amino acid substitutions and
are frequently derived
from cloned genes that have been subjected to site-directed or random
mutagenesis, or from
completely synthetic genes.
[0068] The terms "DNA", "nucleic acid", "nucleic acid molecule",
"polynucleotide" and
the like are used interchangeably herein to refer to a polymeric form of
nucleotides of any length,
either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-
limiting examples of
polynucleotides include linear and circular nucleic acids, messenger RNA
(mRNA),
complementary DNA (cDNA), recombinant polynucleotides, vectors, probes,
primers and the like.
[0069] As used herein in the context of the structure of a polypeptide,
"N-terminus" (or
"amino terminus") and "C-terminus" (or "carboxyl terminus") refer to the
extreme amino and
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carboxyl ends of the polypeptide, respectively, while the terms "N-terminal"
and "C-terminal"
refer to relative positions in the amino acid sequence of the polypeptide
toward the N-terminus
and the C-terminus, respectively, and can include the residues at the N-
terminus and C-terminus,
respectively. "Immediately N-terminal" or "immediately C-terminal" refers to a
position of a first
amino acid residue relative to a second amino acid residue where the first and
second amino acid
residues are covalently bound to provide a contiguous amino acid sequence.
[0070] "Derived from", in the context of an amino acid sequence or
polynucleotide
sequence (e.g., an amino acid sequence "derived from" an IL-10 polypeptide),
is meant to indicate
that the polypeptide or nucleic acid has a sequence that is based on that of a
reference polypeptide
or nucleic acid (e.g., a naturally occurring IL-10 polypeptide or an IL-10-
encoding nucleic acid),
and is not meant to be limiting as to the source or method in which the
protein or nucleic acid is
made. By way of example, the term "derived from" includes homologs or variants
of reference
amino acid or DNA sequences.
[0071] In the context of a polypeptide, the term "isolated" refers to a
polypeptide of
interest that, if naturally occurring, is in an environment different from
that in which it can
naturally occur. "Isolated" is meant to include polypeptides that are within
samples that are
substantially enriched for the polypeptide of interest and/or in which the
polypeptide of interest is
partially or substantially purified. Where the polypeptide is not naturally
occurring, "isolated"
indicates that the polypeptide has been separated from an environment in which
it was made by
either synthetic or recombinant means.
[0072] "Enriched" means that a sample is non-naturally manipulated (e.g.,
by a scientist)
so that a polypeptide of interest is present in a) a greater concentration
(e.g., at least 3-fold greater,
at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or
more) than the
concentration of the polypeptide in the starting sample, such as a biological
sample (e.g., a sample
in which the polypeptide naturally occurs or in which it is present after
administration), or b) a
concentration greater than the environment in which the polypeptide was made
(e.g., as in a
bacterial cell).
[0073] "Substantially pure" indicates that a component (e.g., a
polypeptide) makes up
greater than about 50% of the total content of the composition, and typically
greater than about
60% of the total polypeptide content. More typically, "substantially pure"
refers to compositions
in which at least 75%, at least 85%, at least 90% or more of the total
composition is the
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component of interest. In some cases, the polypeptide will make up greater
than about 90%, or
greater than about 95% of the total content of the composition.
[0074] The terms "specifically binds" or "selectively binds", when
referring to a
ligand/receptor, antibody/antigen, or other binding pair, indicates a binding
reaction which is
determinative of the presence of the protein in a heterogeneous population of
proteins and other
biologics. Thus, under designated conditions, a specified ligand binds to a
particular receptor and
does not bind in a significant amount to other proteins present in the sample.
The antibody, or
binding composition derived from the antigen-binding site of an antibody, of
the contemplated
method binds to its antigen, or a variant or mutein thereof, with an affinity
that is at least two-fold
greater, at least ten times greater, at least 20-times greater, or at least
100-times greater than the
affinity with any other antibody, or binding composition derived therefrom. In
a particular
embodiment, the antibody will have an affinity that is greater than about 109
liters/mol, as
determined by, e.g., Scatchard analysis (Munsen, et al. 1980 Analyt. Biochem.
107:220-239).
IL-10 and PEG-IL-10
[0075] The anti-inflammatory cytokine IL-10, also known as human cytokine
synthesis
inhibitory factor (CSIF), is classified as a type(class)-2 cytokine, a set of
cytokines that includes
IL-19, IL-20, IL-22, IL-24 (Mda-7), and IL-26, interferons (IFN-a, -(3, -y, -
6, -6, -lc, 42, and -T) and
interferon-like molecules (limitin, IL-28A, IL-28B, and IL-29).
[0076] IL-10 is a cytokine with pleiotropic effects in immunoregulation
and inflammation.
It is produced by mast cells, counteracting the inflammatory effect that these
cells have at the site
of an allergic reaction. While it is capable of inhibiting the synthesis of
pro-inflammatory
cytokines such as IFN-y, IL-2, IL-3, TNFa and GM-CSF, IL-10 is also
stimulatory towards certain
T cells and mast cells and stimulates B-cell maturation, proliferation and
antibody production. IL-
can block NF-KB activity and is involved in the regulation of the JAK-STAT
signaling
pathway. It also induces the cytotoxic activity of CD8+ T-cells and the
antibody production of B-
cells, and it suppresses macrophage activity and tumor-promoting inflammation.
The regulation
of CD8+ T-cells is dose-dependent, wherein higher doses induce stronger
cytotoxic responses.
[0077] As indicated elsewhere herein, IL-10 is deemed to be an anti-
inflammatory and
immuno-suppressive cytokine that inhibits the secretion of IFN-y, IL-12
(D'Andrea, A., et al.
(1993) J Exp Med 178(3):1041-48), and TNFa (Armstrong, L., et al. (1996)
Thorax 51(2):143-49).
IL-10 also inhibits antigen presentation and subsequent activation of CD4+ T
cells (de Waal
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Malefyt, R., et al. (1991) J Exp Med 174(5):1209-20; de Waal Malefyt, R., et
al. (1991) J Exp
Med 174(4):915-24) and is thus widely considered to be a potent immune
suppressive cytokine.
[0078] Human IL-10 is a homodimer with a molecular mass of 37kDa, wherein
each
18.5kDa monomer comprises 178 amino acids, the first 18 of which comprise a
signal peptide,
and two cysteine residues that form two intramolecular disulfide bonds. The IL-
10 dimer becomes
biologically inactive upon disruption of the non-covalent interactions between
the two monomer
subunits.
[0079] As alluded to above, the terms "IL-10", "IL-10 polypeptide(s), "IL-
10
molecule(s)", "IL-10 agent(s)" and the like are intended to be broadly
construed and include, for
example, human and non-human IL-10 ¨ related polypeptides, including homologs,
variants
(including muteins), and fragments thereof, as well as IL-10 polypeptides
having, for example, a
leader sequence (e.g., the signal peptide), and modified versions of the
foregoing. The present
disclosure contemplates pegylated forms of human IL-10 (NP 000563) and murine
IL-10
(NP 034678), which exhibit 80% homology, and use thereof. In addition, the
scope of the present
disclosure includes pegylated IL-10 orthologs, and modified forms thereof,
from other mammalian
species, including rat (accession NP 036986.2; GI 148747382); cow (accession
NP 776513.1; GI
41386772); sheep (accession NP 001009327.1; GI 57164347); dog (accession
ABY86619.1; GI
166244598); and rabbit (accession AAC23839.1; GI 3242896).
[0080] The IL-10 receptor, a type II cytokine receptor, consists of alpha
and beta subunits,
which are also referred to as R1 and R2, respectively. Receptor activation
requires binding to both
alpha and beta. One homodimer of an IL-10 polypeptide binds to alpha and the
other homodimer
of the same IL-10 polypeptide binds to beta.
[0081] As used herein, the terms "pegylated IL-10", "PEG-IL-10" and the
like refer to an
IL-10 molecule having one or more polyethylene glycol molecules covalently
attached to at least
one amino acid residue of the IL-10 protein, generally via a linker, such that
the attachment is
stable. The terms "monopegylated IL-10" and "mono-PEG-IL-10" indicate that one
polyethylene
glycol molecule is covalently attached to a single amino acid residue on one
subunit of the IL-10
dimer, generally via a linker. As used herein, the terms "dipegylated IL-10"
and "di-PEG-IL-10"
indicate that at least one polyethylene glycol molecule is attached to a
single residue on each
subunit of the IL-10 dimer, generally via a linker.
[0082] In certain embodiments, the PEG-IL-10 used in the present
disclosure is a mono-
PEG-IL-10 in which one to nine PEG molecules are covalently attached via a
linker to the alpha
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amino group of the amino acid residue at the N-terminus of one subunit of the
IL-10 dimer.
Monopegylation on one IL-10 subunit generally results in a non-homogeneous
mixture of non-
pegylated, monopegylated and dipegylated IL-10 due to subunit shuffling.
Moreover, allowing a
pegylation reaction to proceed to completion will generally result in non-
specific and multi-
pegylated IL-10, thus reducing its bioactivity. Thus, particular embodiments
of the present
disclosure comprise the administration of a mixture of mono- and di-pegylated
IL-10 produced by
the methods described herein.
[0083] In particular embodiments, the average molecular weight of the PEG
moiety is
between about 5kDa and about 50kDa. Although the method or site of PEG
attachment to IL-10
is not critical, in certain embodiments the pegylation does not alter, or only
minimally alters, the
activity of the IL-10 peptide. In certain embodiments, the increase in half-
life is greater than any
decrease in biological activity. The biological activity of PEG-IL-10 is
typically measured by
assessing the levels of inflammatory cytokines (e.g., TNFa or IFNy) in the
serum of subjects
challenged with a bacterial antigen (lipopolysaccharide (LPS)) and treated
with PEG-IL-10, as
described in U.S. Pat. No. 7,052,686.
[0084] IL-10 variants can be prepared with various objectives in mind,
including
increasing serum half-life, reducing an immune response against the IL-10,
facilitating purification
or preparation, decreasing conversion of IL-10 into its monomeric subunits,
improving therapeutic
efficacy, and lessening the severity or occurrence of side effects during
therapeutic use. The
amino acid sequence variants are usually predetermined variants not found in
nature, although
some can be post-translational variants, e.g., glycosylated variants. The
present disclosure
contemplates the use of any pegylated variant of IL-10 provided it retains a
suitable level of IL-10
activity.
[0085] The phrase "conservative amino acid substitution" refers to
substitutions that
preserve the activity of the protein by replacing an amino acid(s) in the
protein with an amino acid
with a side chain of similar acidity, basicity, charge, polarity, or size of
the side chain.
Conservative amino acid substitutions generally entail substitution of amino
acid residues within
the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T,
S; 5) Q, N; and 6) D,
E. Guidance for substitutions, insertions, or deletions can be based on
alignments of amino acid
sequences of different variant proteins or proteins from different species.
Thus, in addition to any
naturally-occurring IL-10 polypeptide, the present disclosure contemplates
having 1, 2, 3, 4, 5, 6,
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7, 8, 9, or 10 usually no more than 20, 10, or 5 amino acid substitutions,
where the substitution is
usually a conservative amino acid substitution.
[0086] The present disclosure also contemplates pegylated forms of active
fragments (e.g.,
subsequences) of mature IL-10 containing contiguous amino acid residues
derived from the
mature IL-10. The length of contiguous amino acid residues of a peptide or a
polypeptide
subsequence varies depending on the specific naturally-occurring amino acid
sequence from
which the subsequence is derived. In general, peptides and polypeptides can be
from about 20
amino acids to about 40 amino acids, from about 40 amino acids to about 60
amino acids, from
about 60 amino acids to about 80 amino acids, from about 80 amino acids to
about 100 amino
acids, from about 100 amino acids to about 120 amino acids, from about 120
amino acids to about
140 amino acids, from about 140 amino acids to about 150 amino acids, from
about 150 amino
acids to about 155 amino acids, from about 155 amino acids up to the full-
length peptide or
polypeptide.
[0087] Additionally, IL-10 polypeptides can have a defined sequence
identity compared to
a reference sequence over a defined length of contiguous amino acids (e.g., a
"comparison
window"). Methods of alignment of sequences for comparison are well-known in
the art. Optimal
alignment of sequences for comparison can be conducted, e.g., by the local
homology algorithm of
Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of
Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity
method of
Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin
Genetics Software Package, Madison, Wis.), or by manual alignment and visual
inspection (see,
e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995
supplement)).
[0088] As an example, a suitable IL-10 polypeptide that can be pegylated
can comprise an
amino acid sequence having at least about 75%, at least about 80%, at least
about 85%, at least
about 90%, at least about 95%, at least about 98%, or at least about 99%,
amino acid sequence
identity to a contiguous stretch of from about 20 amino acids to about 40
amino acids, from about
40 amino acids to about 60 amino acids, from about 60 amino acids to about 80
amino acids, from
about 80 amino acids to about 100 amino acids, from about 100 amino acids to
about 120 amino
acids, from about 120 amino acids to about 140 amino acids, from about 140
amino acids to about
150 amino acids, from about 150 amino acids to about 155 amino acids, from
about 155 amino
acids up to the full-length IL-10 peptide or polypeptide.
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[0089] As discussed further below, the IL-10 polypeptides can be isolated
from a natural
source (e.g., an environment other than its naturally-occurring environment)
and can also be
recombinantly made (e.g., in a genetically modified host cell such as
bacteria, yeast, Pichia, insect
cells, and the like), where the genetically modified host cell is modified
with a nucleic acid
comprising a nucleotide sequence encoding the polypeptide. The IL-10
polypeptides can also be
synthetically produced (e.g., by cell-free chemical synthesis).
[0090] Nucleic acid molecules encoding an IL-10 molecule are contemplated
by the
present disclosure, including their naturally-occurring and non-naturally
occurring isoforms,
allelic variants and splice variants. The present disclosure also encompasses
nucleic acid
sequences that vary in one or more bases from a naturally-occurring DNA
sequence but still
translate into an amino acid sequence that corresponds to an IL-10 polypeptide
due to degeneracy
of the genetic code.
IL-12
[0091] Interleukin-12 (IL-12) is a pleiotropic cytokine naturally
produced by macrophages,
B-lymphoblastoid cells, dendritic cells, and neutrophils in response to
antigenic stimulation. It
was first described as a factor secreted from PMA-induced EBV-transformed B-
cell lines. IL-12
is involved in the differentiation of naïve T cells into Thl cells, can
stimulate the growth and
function of T cells, and mediates enhancement of the cytotoxic activity of NK
cells and CD8+
cytotoxic T lymphocytes. As such, IL-12 activates both innate (NK cells) and
adaptive (cytotoxic
T Lymphocytes). IL-12 also stimulates the production of IFNy and TNFa from T
cells and NK
cells, and reduces IL-4 ¨ mediated suppression of IFNy.
[0092] As indicated elsewhere herein, the terms "IL-12", "IL-12
polypeptide(s)," "IL-12-
agent(s)", "IL-12 molecule(s)" and the like are intended to be construed
broadly and include, for
example, human and non-human IL-12 ¨ related polypeptides, including homologs,
variants
(including muteins), and fragments thereof, as well as IL-12 polypeptides
having, for example, a
leader sequence (e.g., a signal peptide).
[0093] Structurally, IL-12 comprises a complex of four alpha helices. It
is a heterodimeric
cytokine encoded by two separate genes, IL-12, Chain A (p35) and IL-12, Chain
B (p40). Human
IL-12A is a 306 amino acid residue polypeptide (FIG. 1; accession no. 1F45 A),
while human IL-
12B is a 197 amino acid residue polypeptide (FIG. 1; accession no. 1F45 B).
The active
heterodimer (p70) and a homodimer of p40 are formed following protein
synthesis. IL-12 binds to
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the IL-12 receptor, a heterodimeric receptor formed by IL-12R-131 and IL-12R-
132, which initiates
a signaling cascade comprising several transcription factors involved in the
JAK-STAT pathway.
[0094] The present disclosure contemplates active fragments (e.g.,
subsequences) of
mature IL-12 containing contiguous amino acid residues derived from the mature
IL-12. The
length of contiguous amino acid residues of a peptide or a polypeptide
subsequence varies
depending on the specific naturally-occurring amino acid sequence from which
the subsequence is
derived. In general, peptides and polypeptides can be from about 20 amino
acids to about 40
amino acids, from about 40 amino acids to about 60 amino acids, from about 60
amino acids to
about 80 amino acids, from about 80 amino acids to about 100 amino acids, from
about 100 amino
acids to about 120 amino acids, from about 120 amino acids to about 140 amino
acids, from about
140 amino acids to about 160 amino acids, from about 160 amino acids to about
180 amino acids,
from about 180 amino acids to about 190 amino acids, from about 190 amino
acids to about 194
amino acids, from about 194 amino acids to about 196 amino acids, from about
196 amino acids to
about 210 amino acids, from about 210 amino acids to about 230 amino acids,
from about 230
amino acids to about 250 amino acids, from about 250 amino acids to about 270
amino acids, from
about 270 amino acids to about 290 amino acids, from about 290 amino acids to
about 295 amino
acids, from about 295 amino acids to about 300 amino acids, from about 300
amino acids to about
304 amino acids, and from about 304 amino acids to about 306 amino acids.
[0095] Additionally, IL-12 polypeptides can have a defined sequence
identity compared to
a reference sequence over a defined length of contiguous amino acids (e.g., a
"comparison
window"). Methods of alignment of sequences for comparison are well-known in
the art and are
described above. As an example, a suitable IL-12 polypeptide can comprise an
amino acid
sequence having at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at
least about 95%, at least about 98%, or at least about 99%, amino acid
sequence identity to a
contiguous stretch of from about 20 amino acids to about 40 amino acids, from
about 40 amino
acids to about 60 amino acids, from about 60 amino acids to about 80 amino
acids, from about 80
amino acids to about 100 amino acids, from about 100 amino acids to about 120
amino acids, from
about 120 amino acids to about 140 amino acids, from about 140 amino acids to
about 160 amino
acids, from about 160 amino acids to about 180 amino acids, from about 180
amino acids to about
190 amino acids, from about 190 amino acids to about 195 amino acids, from
about 194 amino
acids to about 196 amino acids, from about 196 amino acids to about 210 amino
acids, from about
210 amino acids to about 230 amino acids, from about 230 amino acids to about
250 amino acids,
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from about 250 amino acids to about 270 amino acids, from about 270 amino
acids to about 290
amino acids, from about 290 amino acids to about 295 amino acids, from about
295 amino acids to
about 300 amino acids, from about 300 amino acids to about 304 amino acids,
and from about 304
amino acids to about 306 amino acids.
[0096] As indicated elsewhere herein, the IL-12 polypeptides can be
isolated from a
natural source (e.g., an environment other than its naturally-occurring
environment) and can also
be recombinantly made (e.g., in a genetically modified host cell such as
bacteria, yeast, Pichia,
insect cells, and the like), where the genetically modified host cell is
modified with a nucleic acid
comprising a nucleotide sequence encoding the polypeptide. The IL-12
polypeptides can also be
synthetically produced (e.g., by cell-free chemical synthesis).
[0097] Nucleic acid molecules encoding an IL-12 molecule are contemplated
by the
present disclosure, including their naturally-occurring and non-naturally
occurring isoforms,
allelic variants and splice variants. The present disclosure also encompasses
nucleic acid
sequences that vary in one or more bases from a naturally-occurring DNA
sequence but still
translate into an amino acid sequence that corresponds to an IL-12 polypeptide
due to degeneracy
of the genetic code.
[0098] IFNy has been shown to coordinate natural mechanisms of anticancer
defense
(Jakobisiak, M. et al. (2013) Immunol Lett 90:103-22). By stimulating the
production of IFNy,
IL-12 increases the production of the inducible protein-10 chemokine (IP-10 or
CXCL10), which,
in turn, mediates IL-12's anti-angiogenic effect. Because of its ability to
induce immune
responses and its anti-angiogenic activity, IL-12 has been evaluated as an
oncology therapeutic.
IL-12 may be useful in treating other disorders, including psoriasis and
inflammatory bowel
disease.
[0099] Of note, an anti-IL-12/23p40 neutralizing antibody (ustekinumab)
has been
evaluated in the clinic for the treatment of several immune-mediates
disorders, including psoriasis,
ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, atopic
dermatitis, primary biliary
cirrhosis, sarcoidosis and systemic lupus erythematosus. The most advances
studies were directed
to the treatment of psoriasis. (See, e.g., Teng, M. et al. (2015) Nature
Medicine 21(7):719-29).
[00100] Due to its ability to interconnect the innate and adaptive immune
arms and its
potent stimulation of IFNy production, IL-12 was initially thought to
represent an ideal candidate
for tumor immunotherapy. Indeed, results from early studies in animal models
supported its
potential use as a cancer therapeutic. However, clinical studies wherein IL-12
was administered
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systemically yielded a very narrow therapeutic index and resulted in
substantial immune-related
toxicity due to both significantly increased serum cytokines (primarily IFNy
and TNFa) and
autoimmune hepatitis. Together, these cytokines lead to dose-limiting lethal
toxicities and a
maximally tolerated dose for IL-12 of 0.3 [tg/kg subcutaneously daily (see
Bajetta, E., et al., Clin
Cancer Res, 1998. 4(1): p. 75-85; Motzer, R.J., et al., J Interferon Cytokine
Res, 2001. 21(4): p.
257-63; Cebon, J., et al., Cancer Immun, 2003. 3: p. 7; Younes, A., et al.,
Clin Cancer Res, 2004.
10(16): p. 5432-8; and Anse11, S.M., et al., Blood, 2002. 99(1): p. 67-74). As
a result, systemic
administration of IL-12 in the oncology setting was largely deemed unviable.
[See, e.g., Teng, M.
et al. (2015) Nature Medicine 21(7):719-29].
[00101] In an effort to harness IL-12's anti-tumor effect and avoid its
inherent
shortcomings, alternative approaches to systemic administration have been
explored. Autologous
inactivated tumor cells expressing IL-12 and IL-10 were found to induce
beneficial effects in mice
with colon or mammary tumors and lung metastases (Lopez et al. (2005) J
Immunol 175:5885-
94). Despite this apparent positive effect, the report of this 2005 study did
not result in a
concerted effort to explore IL-10/IL-12 systemic combination therapy ¨ likely
due to the toxicity
issues previously experienced with IL-12 in the clinical setting. As indicated
above, IL-12 ¨
associated toxicities that have been observed include flu-like symptoms (e.g.,
headache, fever,
chills, fatigue and arthromyalgia); hematologic toxicity, including
neutropenia and
thrombocytopenia; and hepatic toxicity, manifested by dose-dependent increases
in transaminases,
hyperbilirubinemia, and hypoalbuminemia. Other IL-12 associated adverse
effects include
inflammation of mucus membranes (e.g., oral mucositis, stomatitis and
colitis), hypotension, renal
impairment and gastrointestinal bleeding. These toxic effects have been
associated with the
secondary production of IFNy and TNFa, as well as other cytokines (e.g., IP-10
and MIG). (See,
e.g., Lasek, et al. (2014) Cancer Immunol Immunother 63:419-35; Xu, et al.
Clinical and
Developmental Immunology, volume 2010, Article ID 832454, 9 pp.); Cebon, J.,
et al., Cancer
Immun, 2003. 3: p. 7).
[00102] Recent efforts to leverage IL-12's potential usefulness in
oncology have taken
different directions. For example, clinical studies have been initiated in
which IL-12 is applied as
an adjuvant in cancer vaccines, in gene therapy including loco regional
injections of IL-12
plasmid, and in the form of tumor-targeting immunocytokines. Other strategies
include co-
administration with Treg cell-depleting antibodies (e.g., an anti-CD25
antibody), antibodies
against immune suppressive signals (e.g., CTLA-4), and anticancer drugs. (See,
e.g., Lasek, et al.
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(2014) Cancer Immunol Immunother 63:419-35; Xu, etal. Clinical and
Developmental
Immunology, volume 2010, Article ID 832454, 9 pp.)). These approaches make it
further
apparent that the oncology community has concluded that IL-10/IL-12 systemic
combination
therapy is intractable.
Co-administration of IL-10 and IL-12
[00103] The combination of PEG-IL-10 and IL-12 is thought to exhibit at
least additive,
and possibly synergistic, anti-tumor efficacy. However, the toxicity observed
with IL-12
monotherapy has heretofore limited the exploration of such combination therapy
in human
subjects. In particular, IL-12 exhibits potent immunostimulatory biology which
limits its
maximally tolerated dose (which has been described as 0.5 ¨ 1.25 i.tg/kg; see
Cebon, J., et al.,
Cancer Immun, 2003. 3: p. 7) to an amount which is lower than its maximally
efficacious dose.
Although an understanding of the mechanism underlying this phenomenon is not
required in order
to practice the present disclosure, it is thought to be due to the activation
by IL-12 of both antigen-
non-specific naïve CD4+ and CD8+ T cells and antigen-specific CD4+ and CD8+ T
cells, as well
as NK cells. While IL-12 exhibits a broad spectrum immune stimulation that is
both antigen-
specific and antigen-non-specific, PEG-IL-10 exposure only activates the
antigen-specific
population of CD8+ T cells. As indicated below, when combined, PEG-IL-10
likely limits the
non-antigen-specific immune stimulation of IL-12 and focuses IL-12's
immunostimulatory effects
into the antigen specific, adaptive CD8+ T cell arm of the immune system.
[00104] IL-12's immunostimulatory response comprises, in part, the
induction of IFNy and
TNFa secretion from these cells, and this elevation of serum IFNy, and to a
lesser extent serum
TNFa, is correlated with the onset of immune-related toxicities. Although PEG-
IL-10 treatment
also leads to the elevation of serum IFNy levels, its MTD has not been
established due to
therapeutic benefit obtained at doses ranging from 5-40 pg/kg dosed
subcutaneously daily.
[00105] As described in detail in the Experimental section, the
combinatorial effect of PEG-
IL-10 and IL-12 on tumor size in a murine 4T1 tumor model was evaluated. As
indicated in FIG.
2, administration of combinations of PEG-rMuIL-10 and rMuIL-12 resulted in a
larger reduction
in tumor weight than that observed following administration of either agent
alone. These data
represent the beneficial anti-tumor effects of combination therapy.
[00106] Next, the serum levels IFNy and TNFa induced by PEG-rMuIL-10 and
rMuIL-12,
either alone or in combination, in 4T1 tumor bearing mice were evaluated. The
data indicate that
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exposure to both PEG-rMuIL-10 and rMuIL-12 individually lead to the induction
of serum IFNy
and TNFa. Surprisingly, when combined, administration of IL-12 and PEG-rMuIL-
10 resulted in
lower IFNy (FIG. 3A) and TNFa (FIG. 3B) serum levels. In particular, combined
exposure of IL-
12 and PEG-rMuIL-10 exhibited significantly lower serum IFNy than IL-12 alone.
A particular
embodiment of the present disclosure comprises the use of PEG-IL-10 to lower
the serum
cytokine (IFNy and TNFa) levels induced by IL-12 treatment in order to
detoxify IL-12, while
simultaneously enhancing the anti-tumor benefits of administering the two
agents together.
[00107] As noted above, while recent reports relating to combinations of
IL-10 and IL-12 in
the immunoncology setting have reported potential synergistic anti-tumor
function, there is no
disclosure of the potential control of IL-12 ¨ mediated toxicity by such
combination therapy.
Therefore, the data and other findings reported herein that the combination of
IL-12 and PEG-IL-
results in control of IL-12 ¨ associated toxicity, as indicated by a
significant decrease in serum
IFNy (relative to that observed following IL-12 monotherapy) was both
surprising and
unexpected.
Serum Concentrations
[00108] The blood plasma levels of IL-10 in the methods described herein
can be
characterized in several manners, including: (1) a mean IL-10 serum trough
concentration above
some specified level or in a range of levels; (2) a mean IL-10 serum trough
concentration above
some specified level for some amount of time; (3) a steady state IL-10 serum
concentration level
above or below some specified level or in a range of levels; or (4) a C. of
the concentration
profile above or below some specified level or in some range of levels. As set
forth herein, mean
serum trough IL-10 concentrations have been found to be of particular import
for efficacy in
certain indications. Blood plasma levels of IL-12 can be characterized in a
similar manner.
[00109] As set forth above, the desired IL-10 serum trough concentration
may depend on a
number of factors, including the nature of the disease, disorder or condition
(e.g., localized tumor
or metastatic disease), the extent to which the subject is suffering from the
malady (e.g., early
versus late stage disease), whether combination therapy is being administered,
and patient-specific
parameters (e.g., hepatic and renal function). By way of example, co-
administration of PEG-IL-10
and a chemotherapeutic agent may only require a serum trough in the ¨1-2 ng/mL
range in order
to observe clinical benefit, while metastatic cancer may require 6-10 ng/mL or
more to achieve
comparable clinical benefit.
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[00110] In particular embodiments of the present disclosure, the mean IL-
10 serum trough
concentration is at least 6.0 ng/mL, at least 7.0 ng/mL, at least 8.0 ng/mL,
and least 9.0 ng/mL, at
least 10.0 ng/mL, at least 11.0 ng/mL, at least 12.0 ng/mL, at least 13.0
ng/mL, at least 14.0
ng/mL, at least 15.0 ng/mL, at least 16.0 ng/mL, at least 17.0 ng/mL, at least
18.0 ng/mL, at least
19.0 ng/mL, at least 20.0 ng/mL, at least 21.0 ng/mL, at least 22.0 ng/mL, or
greater than 22.0
ng/mL.
[00111] In other particular embodiments, the mean IL-10 serum trough
concentration is at
least 1.0 ng/mL, at least 1.5 ng/mL, at least 2.0 ng/mL, at least 2.5 ng/mL,
at least 3.0 ng/mL, at
least 3.5 ng/mL, at least 4.0 ng/mL, at least 4.5 ng/mL, at least 5.0 ng/mL,
and least 5.5 ng/mL, at
least 6.0 ng/mL, at least 6.5 ng/mL or greater than 7 ng/mL.
[00112] In further embodiments, the period of time is at least 12 hours,
at least 24 hours, at
least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least
3 weeks, at least 1
month, at least 6 weeks, at least 2 months, at least 3 months, or greater than
3 months.
[00113] In particular embodiments of the present disclosure, the mean IL-
10 serum trough
concentration is maintained for at least 85% of the period of time, at least
90%, at least 95%, at
least 98%, at least 99% or 100% of the period of time.
[00114] In still further embodiments of the present disclosure, blood
plasma and/or serum
level concentration profiles that can be produced include: a mean IL-10 plasma
and/or serum
trough concentration of greater than about 1.0 pg/mL, greater than about 10.0
pg/mL, greater than
about 20.0 pg/mL, greater than about 30 pg/mL, greater than about 40 pg/mL,
greater than about
50.0 pg/mL, greater than about 60.0 pg/mL, greater than about 70.0 pg/mL,
greater than about
80.0 pg/mL, greater than about 90 pg/mL, greater than about 0.1 ng/mL, greater
than about 0.2
ng/mL, greater than about 0.3 ng/mL, greater than about 0.4 ng/mL, greater
than about 0.5 ng/mL,
greater than about 0.6 ng/mL, greater than about 0.7 ng/mL, greater than about
0.8 ng/mL, greater
than about 0.9 ng/mL, greater than about 1.0 ng/mL, greater than about 1.5
ng/mL, greater than
about 2.0 ng/mL, greater than about 2.5 ng/mL, greater than about 3.0 ng/mL,
greater than about
3.5 ng/mL, greater than about 4.0 ng/mL, greater than about 4.5 ng/mL, greater
than about 5.0
ng/mL, greater than about 5.5 ng/mL, greater than about 6.0 ng/mL, greater
than about 6.5 ng/mL,
greater than about 7.0 ng/mL, greater than about 7.5 ng/mL, greater than about
8.0 ng/mL, greater
than about 8.5 ng/mL, greater than about 9.0 ng/mL, greater than about 9.5
ng/mL, or greater than
about 10.0 ng/mL.
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[00115] In particular embodiments of the present disclosure, a mean IL-10
serum trough
concentration is in the range of from 1.0 pg/mL to 10 ng/mL. In some
embodiments, the mean IL-
serum trough concentration is in the range of from 1.0 pg/mL to 100 pg/mL. In
other
embodiments, the mean IL-10 serum trough concentration is in the range of from
0.1 ng/mL to 1.0
ng/mL. In still other embodiments, the mean IL-10 serum trough concentration
is in the range of
from 1.0 ng/mL to 10 ng/mL. It is to be understood that the present disclosure
contemplates
ranges incorporating any concentrations encompassed by those set forth herein
even if such ranges
are not explicitly recited. By way of example, the mean serum IL-10
concentration in an
embodiment can be in the range of from 0.5 ng/mL to 5 ng/mL. By way of further
examples,
particular embodiments of the present disclosure comprise a mean IL-10 serum
trough
concentration in a range of from about 0.5 ng/mL to about 10.5 ng/mL, from
about 1.0 ng/mL to
about 10.0 ng/mL, from about 1.0 ng/mL to about 9.0 ng/mL, from about 1.0
ng/mL to about 8.0
ng/mL, from about 1.0 ng/mL to about 7.0 ng/mL, from about 1.5 ng/mL to about
10.0 ng/mL,
from about 1.5 ng/mL to about 9.0 ng/mL, from about 1.5 ng/mL to about 8.0
ng/mL, from about
1.5 ng/mL to about 7.0 ng/mL, from about 2.0 ng/mL to about 10.0 ng/mL, from
about 2.0 ng/mL
to about 9.0 ng/mL, from about 2.0 ng/mL to about 8.0 ng/mL, and from about
2.0 ng/mL to about
7.0 ng/mL.
[00116] In particular embodiments, a mean IL-10 serum trough concentration
of 1 - 2
ng/mL is maintained over the duration of treatment. The present disclosure
also contemplates
embodiments wherein the mean IL-10 serum peak concentration is less than or
equal to about 10.0
ng/mL over the duration of treatment. Further embodiments contemplate a mean
IL-10 serum
trough concentration greater than or equal to about 10.0 ng/mL. The optimal
mean serum
concentration is generally that at which the desired therapeutic effect is
achieved without
introducing undesired adverse effects.
[00117] Certain embodiments of the present disclosure provide a method for
monitoring a
subject receiving IL-10 therapy to predict, and thus potentially avoid,
adverse effects, the method
comprising: (1) measuring the subject's peak concentration of IL-10; (2)
measuring the subject's
trough concentration of IL-10; (3) calculating a peak-trough fluctuation; and,
(4) using the
calculated peak-trough fluctuation to predict potential adverse effects in the
subject. In particular
subject populations, a smaller peak-trough fluctuation indicates a lower
probability that the subject
will experience IL-10 - related adverse effects. In addition, in some
embodiments particular peak-
trough fluctuations are determined for the treatment of particular diseases,
disorders and
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conditions using particular dosing parameters, and those fluctuations are used
as reference
standards.
[00118] For the majority of drugs, plasma drug concentrations decline in a
multi-
exponential fashion. Immediately after intravenous administration, the drug
rapidly distributes
throughout an initial space (minimally defined as the plasma volume), and then
a slower,
equilibrative distribution to extravascular spaces (e.g., certain tissues)
occurs. Intravenous IL-10
administration is associated with such a two-compartment kinetic model (see
Rachmawati, H. et
al. (2004) Pharm. Res. 21(11):2072-78). The pharmacokinetics of subcutaneous
recombinant hIL-
has also been studied (Radwanski, E. et al. (1998) Pharm. Res. 15(12):1895-
1901). Thus,
volume-of-distribution considerations are pertinent when assessing appropriate
IL-10 dosing-
related parameters. Moreover, efforts to target IL-10 agents to specific cell
types have been
explored (see, e.g., Rachmawati, H. (May 2007) Drug Met. Dist. 35(5):814-21),
and the
leveraging of IL-10 pharmacokinetic and dosing principles can prove invaluable
to the success of
such efforts.
[00119] The present disclosure contemplates administration of any dose and
dosing regimen
that results in maintenance of any of the IL-10 serum trough concentrations
set forth above. By
way of example, but not limitation, when the subject is a human, non-pegylated
hIL-10 can be
administered at a dose greater than 0.5 g/kg/day, greater than 1.0 g/kg/day,
greater than 2.5
g/kg/day, greater than 5 g/kg/day, greater than 7.5 g/kg, greater than 10.0
[tg/kg, greater than
12.5 [tg/kg, greater than 15 [tg/kg/day, greater than 17.5 g/kg/day, greater
than 20 g/kg/day,
greater than 22.5 g/kg/day, greater than 25 [tg/kg/day, greater than 30
g/kg/day, or greater than
35 g/kg/day. In addition, by way of example, but not limitation, when the
subject is a human,
pegylated hIL-10 comprising a relatively small PEG (e.g., 5kDa mono- di-PEG-
hIL-10) can be
administered at a dose greater than 0.5 [tg/kg/day, greater than 0.75
g/kg/day, greater than 1.0
[tg/kg/day, greater than 1.25 g/kg/day, greater than 1.5 [tg/kg/day, greater
than 1.75 [tg/kg/day,
greater than 2.0 g/kg/day, greater than 2.25 [tg/kg/day, greater than 2.5
[tg/kg/day, greater than
2.75 [tg/kg/day, greater than 3.0 g/kg/day, greater than 3.25 [tg/kg/day,
greater than 3.5
[tg/kg/day, greater than 3.75 g/kg/day, greater than 4.0 [tg/kg/day, greater
than 4.25 [tg/kg/day,
greater than 4.5 g/kg/day, greater than 4.75 [tg/kg/day, or greater than 5.0
[tg/kg/day.
[00120] The skilled artisan (e.g., a pharmacologist) is able to determine
the optimum dosing
regimen(s) when a PEG-IL-10 is administered in combination with an IL-12
agent. By way of
example, in some embodiments the optimum PEG-IL-10 dosing regimen may require
a reduction
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in the amount of PEG-IL-10 administered per dose (e.g., less than 1.0
tg/kg/day, less than 0.75
g/kg/day, less than 0.5 tg/kg/day, less than 0.25 g/kg/day, or less than
0.125 g/kg/day). In
certain exemplary embodiments of the present disclosure, a mean IL-10 serum
trough
concentration may be in a range of from about 0.1 ng/mL to about 9.5 ng/mL,
from about 0.25
ng/mL to about 8.0 ng/mL, from about 0.5 ng/mL to about 7.0 ng/mL, from about
0.75 ng/mL to
about 6.0 ng/mL, or from about 1.0 ng/mL to about 5.0 ng/mL.
[00121] The present disclosure contemplates dosing an IL-12 agent such
that the serum
concentration achieves a peak and is then cleared such that it is essentially
unmeasurable before it
is administered again. By way of example, when a PEG-IL-10 is administered
every 24 hours to
maintain a serum trough concentration of ¨ 10 ng/mL, an IL-12 agent can be co-
administered in
an amount (e.g., 5 pg/kg/day) that results in a peak less that its MTD and
then is metabolized such
that there is no measurable serum level at the end of a 24-hour dosing cycle.
As with
administration of a PEG-IL-10, the dose of an IL-12 agent may depend on a
number of factors,
including the nature of the disease, disorder or condition (e.g., localized
tumor or metastatic
disease), the extent to which the subject is suffering from the malady (e.g.,
early versus late stage
disease), whether combination therapy is being administered, and patient-
specific parameters (e.g.,
hepatic and renal function).
[00122] When a PEG-IL-10 is administered in combination with an IL-12
agent such as
those described herein, one or more of the dosing parameters of the PEG-IL-10
applicable to
monotherapy can be modified while the dosing parameters of the IL-12 agent
applicable to
monotherapy can remain the same; one or more of the dosing parameters of the
PEG-IL-10
applicable to monotherapy can remain the same while one or more of the dosing
parameters of the
IL-12 agent applicable to monotherapy can be modified; one or more of the
dosing parameters of
the PEG-IL-10 and the IL-12 agent applicable to monotherapy can be modified;
or the dosing
parameters of each of the PEG-IL-10 and the IL-12 agent can remain the same.
Methods of Production of IL-10
[00123] A polypeptide of the present disclosure can be produced by any
suitable method,
including non-recombinant (e.g., chemical synthesis) and recombinant methods.
A. Chemical Synthesis
[00124] Where a polypeptide is chemically synthesized, the synthesis can
proceed via
liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS) allows the
incorporation of
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unnatural amino acids and/or peptide/protein backbone modification. Various
forms of SPPS,
such as 9-fluorenylmethoxycarbonyl (Fmoc) and t-butyloxycarbonyl (Boc), are
available for
synthesizing polypeptides of the present disclosure. Details of the chemical
syntheses are known
in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero
J.A. et al., (2005)
Protein Pept Lett. 12:723-8).
[00125] Solid phase peptide synthesis can be performed as described
hereafter. The alpha
functions (Na) and any reactive side chains are protected with acid-labile or
base-labile groups.
The protective groups are stable under the conditions for linking amide bonds
but can readily be
cleaved without impairing the peptide chain that has formed. Suitable
protective groups for the a-
amino function include, but are not limited to, the following: Boc,
benzyloxycarbonyl (Z), 0-
chlorbenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl, tert-amyloxycarbonyl
(Amoc), a, a-
dimethy1-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl, 2-cyano-t-butoxy-
carbonyl, Fmoc, 1-
(4,4-dimethy1-2,6-dioxocylohex-1-ylidene)ethyl (Dde) and the like.
[00126] Suitable side chain protective groups include, but are not limited
to: acetyl, allyl
(All), allyloxycarbonyl (Alloc), benzyl (Bzl), benzyloxycarbonyl (Z), t-
butyloxycarbonyl (Boc),
benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl (tBu), t-
butyldimethylsilyl, 2-
chlorobenzyl, 2-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyl, cyclohexyl,
cyclopentyl,
dimethy1-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), isopropyl, 4-methoxy-2,3-6-
trimethylbenzylsulfonyl (Mtr), 2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc),
pivalyl,
tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl, trimethylsilyl and
trityl (Trt).
[00127] In the solid phase synthesis, the C-terminal amino acid is coupled
to a suitable
support material. Suitable support materials are those which are inert towards
the reagents and
reaction conditions for the step-wise condensation and cleavage reactions of
the synthesis process
and which do not dissolve in the reaction media being used. Examples of
commercially-available
support materials include styrene/divinylbenzene copolymers which have been
modified with
reactive groups and/or polyethylene glycol; chloromethylated
styrene/divinylbenzene copolymers;
hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers; and
the like. When
preparation of the peptidic acid is desired, polystyrene (1%)-divinylbenzene
or TentaGel
derivatized with 4-benzyloxybenzyl-alcohol (Wang-anchor) or 2-chlorotrityl
chloride can be used.
In the case of the peptide amide, polystyrene (1%) divinylbenzene or TentaGel
derivatized with
5-(4'-aminomethyl)-3',5'-dimethoxyphenoxy)valeric acid (PAL-anchor) or p-(2,4-
dimethoxyphenyl-amino methyl)-phenoxy group (Rink amide anchor) can be used.
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[00128] The linkage to the polymeric support can be achieved by reacting
the C-terminal
Fmoc-protected amino acid with the support material by the addition of an
activation reagent in
ethanol, acetonitrile, N,N-dimethylformamide (DMF), dichloromethane,
tetrahydrofuran, N-
methylpyrrolidone or similar solvents at room temperature or elevated
temperatures (e.g., between
40 C and 60 C) and with reaction times of, e.g., 2 to 72 hours.
[00129] The coupling of the Na-protected amino acid (e.g., the Fmoc amino
acid) to the
PAL, Wang or Rink anchor can, for example, be carried out with the aid of
coupling reagents such
as N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or
other
carbodiimides, 2-(1H-benzotriazol-1-y1)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU) or
other uronium salts, 0-acyl-ureas, benzotriazol-1-yl-tris-pyrrolidino-
phosphonium
hexafluorophosphate (PyBOP) or other phosphonium salts, N-hydroxysuccinimides,
other N-
hydroxyimides or oximes in the presence or absence of 1-hydroxybenzotriazole
or 1-hydroxy-7-
azabenzotriazole, e.g., with the aid of TBTU with addition of HOBt, with or
without the addition
of a base such as, for example, diisopropylethylamine (DIEA), triethylamine or
N-
methylmorpholine, e.g., diisopropylethylamine with reaction times of 2 to 72
hours (e.g., 3 hours
in a 1.5 to 3-fold excess of the amino acid and the coupling reagents, for
example, in a 2-fold
excess and at temperatures between about 10 C and 50 C, for example, 25 C in a
solvent such as
dimethylformamide, N-methylpyrrolidone or dichloromethane, e.g.,
dimethylformamide).
[00130] Instead of the coupling reagents, it is also possible to use the
active esters (e.g.,
pentafluorophenyl, p-nitrophenyl or the like), the symmetric anhydride of the
Na-Fmoc-amino
acid, its acid chloride or acid fluoride, under the conditions described
above.
[00131] The Na-protected amino acid (e.g., the Fmoc amino acid) can be
coupled to the 2-
chlorotrityl resin in dichloromethane with the addition of DIEA and having
reaction times of 10 to
120 minutes, e.g., 20 minutes, but is not limited to the use of this solvent
and this base.
[00132] The successive coupling of the protected amino acids can be
carried out according
to conventional methods in peptide synthesis, typically in an automated
peptide synthesizer. After
cleavage of the Na-Fmoc protective group of the coupled amino acid on the
solid phase by
treatment with, e.g., piperidine (10% to 50%) in dimethylformamide for 5 to 20
minutes, e.g., 2 x
2 minutes with 50% piperidine in DMF and 1 x 15 minutes with 20% piperidine in
DMF, the next
protected amino acid in a 3 to 10-fold excess, e.g., in a 10-fold excess, is
coupled to the previous
amino acid in an inert, non-aqueous, polar solvent such as dichloromethane,
DMF or mixtures of
the two and at temperatures between about 10 C and 50 C, e.g., at 25 C. The
previously
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mentioned reagents for coupling the first Na-Fmoc amino acid to the PAL, Wang
or Rink anchor
are suitable as coupling reagents. Active esters of the protected amino acid,
or chlorides or
fluorides or symmetric anhydrides thereof can also be used as an alternative.
[00133] At the end of the solid phase synthesis, the peptide is cleaved
from the support
material while simultaneously cleaving the side chain protecting groups.
Cleavage can be carried
out with trifluoroacetic acid or other strongly acidic media with addition of
5%-20% V/V of
scavengers such as dimethyl sulfide, ethylmethyl sulfide, thioani sole,
thiocresol, m-cresol, ani sole
ethanedithiol, phenol or water, e.g., 15% v/v dimethylsulfide/ethanedithiol/m-
cresol 1:1:1, within
0.5 to 3 hours, e.g., 2 hours. Peptides with fully protected side chains are
obtained by cleaving the
2-chlorotrityl anchor with glacial acetic
acid/trifluoroethanol/dichloromethane 2:2:6. The
protected peptide can be purified by chromatography on silica gel. If the
peptide is linked to the
solid phase via the Wang anchor and if it is intended to obtain a peptide with
a C-terminal
alkylamidation, the cleavage can be carried out by aminolysis with an
alkylamine or
fluoroalkylamine. The aminolysis is carried out at temperatures between about -
10 C and 50 C
(e.g., about 25 C), and reaction times between about 12 and 24 hours (e.g.,
about 18 hours). In
addition, the peptide can be cleaved from the support by re-esterification,
e.g., with methanol.
[00134] The acidic solution that is obtained can be admixed with a 3 to 20-
fold amount of
cold ether or n-hexane, e.g., a 10-fold excess of diethyl ether, in order to
precipitate the peptide
and hence to separate the scavengers and cleaved protective groups that remain
in the ether. A
further purification can be carried out by re-precipitating the peptide
several times from glacial
acetic acid. The precipitate that is obtained can be taken up in water or tert-
butanol or mixtures of
the two solvents, e.g., a 1:1 mixture of tert-butanol/water, and freeze-dried.
[00135] The peptide obtained can be purified by various chromatographic
methods,
including ion exchange over a weakly basic resin in the acetate form;
hydrophobic adsorption
chromatography on non-derivatized polystyrene/divinylbenzene copolymers (e.g.,
Amberlite
XAD); adsorption chromatography on silica gel; ion exchange chromatography,
e.g., on
carboxymethyl cellulose; distribution chromatography, e.g., on Sephadex G-25;
countercurrent
distribution chromatography; or high pressure liquid chromatography (HPLC)
e.g., reversed-phase
HPLC on octyl or octadecylsilylsilica (ODS) phases.
B. Recombinant Production
[00136] Methods describing the preparation of human and mouse IL-10 can be
found in, for
example, U.S. Patent No. 5,231,012, which teaches methods for the production
of proteins having
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IL-10 activity, including recombinant and other synthetic techniques. IL-10
can be of viral origin,
and the cloning and expression of a viral IL-10 from Epstein Barr virus (BCRF1
protein) is
disclosed in Moore et al., (1990) Science 248:1230. IL-10 can be obtained in a
number of ways
using standard techniques known in the art, such as those described herein.
Recombinant human
IL-10 is also commercially available, e.g., from PeproTech, Inc., Rocky Hill,
N.J.
[00137] Where a polypeptide is produced using recombinant techniques, the
polypeptide
can be produced as an intracellular protein or as a secreted protein, using
any suitable construct
and any suitable host cell, which can be a prokaryotic or eukaryotic cell,
such as a bacterial (e.g.,
E. coli) or a yeast host cell, respectively. Other examples of eukaryotic
cells that can be used as
host cells include insect cells, mammalian cells, and/or plant cells. Where
mammalian host cells
are used, they can include human cells (e.g., HeLa, 293, H9 and Jurkat cells);
mouse cells (e.g.,
NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos 7 and CV1);
and hamster cells
(e.g., Chinese hamster ovary (CHO) cells).
[00138] A variety of host-vector systems suitable for the expression of a
polypeptide can be
employed according to standard procedures known in the art. See, e.g.,
Sambrook et al., 1989
Current Protocols in Molecular Biology Cold Spring Harbor Press, New York; and
Ausubel et al.
1995 Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for
introduction of
genetic material into host cells include, for example, transformation,
electroporation, conjugation,
calcium phosphate methods and the like. The method for transfer can be
selected so as to provide
for stable expression of the introduced polypeptide-encoding nucleic acid. The
polypeptide-
encoding nucleic acid can be provided as an inheritable episomal element
(e.g., a plasmid) or can
be genomically integrated. A variety of appropriate vectors for use in
production of a polypeptide
of interest are commercially available.
[00139] Vectors can provide for extrachromosomal maintenance in a host
cell or can
provide for integration into the host cell genome. The expression vector
provides transcriptional
and translational regulatory sequences, and can provide for inducible or
constitutive expression
where the coding region is operably-linked under the transcriptional control
of the transcriptional
initiation region, and a transcriptional and translational termination region.
In general, the
transcriptional and translational regulatory sequences can include, but are
not limited to, promoter
sequences, ribosomal binding sites, transcriptional start and stop sequences,
translational start and
stop sequences, and enhancer or activator sequences. Promoters can be either
constitutive or
inducible, and can be a strong constitutive promoter (e.g., T7).
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[00140] Expression constructs generally have convenient restriction sites
located near the
promoter sequence to provide for the insertion of nucleic acid sequences
encoding proteins of
interest. A selectable marker operative in the expression host can be present
to facilitate selection
of cells containing the vector. Moreover, the expression construct can include
additional elements.
For example, the expression vector can have one or two replication systems,
thus allowing it to be
maintained in organisms, for example, in mammalian or insect cells for
expression and in a
prokaryotic host for cloning and amplification. In addition, the expression
construct can contain a
selectable marker gene to allow the selection of transformed host cells.
Selectable genes are well
known in the art and will vary with the host cell used.
[00141] Isolation and purification of a protein can be accomplished
according to methods
known in the art. For example, a protein can be isolated from a lysate of
cells genetically
modified to express the protein constitutively and/or upon induction, or from
a synthetic reaction
mixture by immunoaffinity purification, which generally involves contacting
the sample with an
anti- protein antibody, washing to remove non-specifically bound material, and
eluting the
specifically bound protein. The isolated protein can be further purified by
dialysis and other
methods normally employed in protein purification. In one embodiment, the
protein can be
isolated using metal chelate chromatography methods. Proteins can contain
modifications to
facilitate isolation.
[00142] The polypeptides can be prepared in substantially pure or isolated
form (e.g., free
from other polypeptides). The polypeptides can be present in a composition
that is enriched for
the polypeptide relative to other components that can be present (e.g., other
polypeptides or other
host cell components). For example, purified polypeptide can be provided such
that the
polypeptide is present in a composition that is substantially free of other
expressed proteins, e.g.,
less than about 90%, less than about 60%, less than about 50%, less than about
40%, less than
about 30%, less than about 20%, less than about 10%, less than about 5%, or
less than about 1%.
[00143] An IL-10 polypeptide can be generated using recombinant techniques
to
manipulate different IL-10 ¨ related nucleic acids known in the art to provide
constructs capable
of encoding the IL-10 polypeptide. It will be appreciated that, when provided
a particular amino
acid sequence, the ordinary skilled artisan will recognize a variety of
different nucleic acid
molecules encoding such amino acid sequence in view of her background and
experience in, for
example, molecular biology.
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Amide Bond Substitutions
[00144] In some cases, IL-10 includes one or more linkages other than
peptide bonds, e.g.,
at least two adjacent amino acids are joined via a linkage other than an amide
bond. For example,
in order to reduce or eliminate undesired proteolysis or other means of
degradation, and/or to
increase serum stability, and/or to restrict or increase conformational
flexibility, one or more
amide bonds within the backbone of IL-10 can be substituted.
[00145] In another example, one or more amide linkages (-CO-NH-) in IL-10
can be
replaced with a linkage which is an isostere of an amide linkage, such as -
CH2NH-, -CH2S-, -
CH2CH2-, -CH=CH-(cis and trans), -COCH2-, -CH(OH)CH2- or -CH2S0-. One or more
amide
linkages in IL-10 can also be replaced by, for example, a reduced isostere
pseudopeptide bond.
See Couder et al. (1993) Int. J. Peptide Protein Res. 41:181-184. Such
replacements and how to
effect them are known to those of ordinary skill in the art.
Amino Acid Substitutions
[00146] One or more amino acid substitutions can be made in an IL-10
polypeptide. The
following are non-limiting examples:
[00147] a) substitution of alkyl-substituted hydrophobic amino acids,
including alanine,
leucine, isoleucine, valine, norleucine, (S)-2-aminobutyric acid, (5)-
cyclohexylalanine or other
simple alpha-amino acids substituted by an aliphatic side chain from C1-C10
carbons including
branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions;
[00148] b) substitution of aromatic-substituted hydrophobic amino acids,
including
phenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine, 1-
naphthylalanine, 2-
naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine,
including amino,
alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or
alkoxy (from C1-
C4)-substituted forms of the above-listed aromatic amino acids, illustrative
examples of which are:
2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-
methylphenylalanine,
2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-
methoxytryptophan, 2'-, 3'-,
or 4'-amino-, 2'-, 3'-, or 4'-chloro-, 2, 3, or 4-biphenylalanine, 2'-, 3'-,
or 4'-methyl-, 2-, 3- or 4-
biphenylalanine, and 2- or 3-pyridylalanine;
[00149] c) substitution of amino acids containing basic side chains,
including arginine,
lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine,
including alkyl, alkenyl, or
aryl-substituted (from C1-C10 branched, linear, or cyclic) derivatives of the
previous amino acids,
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whether the substituent is on the heteroatoms (such as the alpha nitrogen, or
the distal nitrogen or
nitrogens, or on the alpha carbon, in the pro-R position for example.
Compounds that serve as
illustrative examples include: N-epsilon-isopropyl-lysine, 3-(4-
tetrahydropyridy1)-glycine, 3-(4-
tetrahydropyridy1)-alanine, N,N-gamma, gamma'-diethyl-homoarginine. Included
also are
compounds such as alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic
acid, alpha-
methyl-histidine, alpha-methyl-ornithine where the alkyl group occupies the
pro-R position of the
alpha-carbon. Also included are the amides formed from alkyl, aromatic,
heteroaromatic (where
the heteroaromatic group has one or more nitrogens, oxygens or sulfur atoms
singly or in
combination), carboxylic acids or any of the many well-known activated
derivatives such as acid
chlorides, active esters, active azolides and related derivatives, and lysine,
ornithine, or 2,3-
diaminopropionic acid;
[00150] d) substitution of acidic amino acids, including aspartic acid,
glutamic acid,
homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl
sulfonamides of 2,4-
diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl
amino acids;
[00151] e) substitution of side chain amide residues, including
asparagine, glutamine, and
alkyl or aromatic substituted derivatives of asparagine or glutamine; and
[00152] f) substitution of hydroxyl-containing amino acids, including
serine, threonine,
homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted
derivatives of serine or
threonine.
[00153] In some cases, IL-10 comprises one or more naturally occurring non-
genetically
encoded L-amino acids, synthetic L-amino acids, or D-enantiomers of an amino
acid. For
example, IL-10 can comprise only D-amino acids. For example, an IL-10
polypeptide can
comprise one or more of the following residues: hydroxyproline, 13-alanine, o-
aminobenzoic acid,
m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3-
diaminopropionic
acid, a-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine,
citrulline, t-butylalanine, t-
butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine,
norleucine, naphthylalanine,
pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine, 2-
fluorophenylalanine, 3-
fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-
tetrahydroisoquinoline-3-
carboxylic acid, 3-2-thienylalanine, methionine sulfoxide, homoarginine, N-
acetyl lysine, 2,4-
diamino butyric acid, rho-aminophenylalanine, N-methylvaline, homocysteine,
homoserine, E-
amino hexanoic acid, w-aminohexanoic acid, w-aminoheptanoic acid, w-
aminooctanoic acid, w-
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aminodecanoic acid, w-aminotetradecanoic acid, cyclohexylalanine, a,y-
diaminobutyric acid, a,13-
diaminopropionic acid, 6-amino valeric acid, and 2,3-diaminobutyric acid.
Additional modifications
[00154] A cysteine residue or a cysteine analog can be introduced into an
IL-10 polypeptide
to provide for linkage to another peptide via a disulfide linkage or to
provide for cyclization of the
IL-10 polypeptide. Methods of introducing a cysteine or cysteine analog are
known in the art; see,
e.g., U.S. Patent No. 8,067,532.
[00155] An IL-10 polypeptide can be cyclized. One or more cysteines or
cysteine analogs
can be introduced into an IL-10 polypeptide, where the introduced cysteine or
cysteine analog can
form a disulfide bond with a second introduced cysteine or cysteine analog.
Other means of
cyclization include introduction of an oxime linker or a lanthionine linker;
see, e.g., U.S. Patent
No. 8,044,175. Any combination of amino acids (or non-amino acid moieties)
that can form a
cyclizing bond can be used and/or introduced. A cyclizing bond can be
generated with any
combination of amino acids (or with an amino acid and -(CH2)õ-00- or -(CH2)õ-
C6H4-00-) with
functional groups which allow for the introduction of a bridge. Some examples
are disulfides,
disulfide mimetics such as the -(CH2)õ- carba bridge, thioacetal, thioether
bridges (cystathionine
or lanthionine) and bridges containing esters and ethers. In these examples, n
can be any integer,
but is frequently less than ten.
[00156] Other modifications include, for example, an N-alkyl (or aryl)
substitution
(v[CONR]), or backbone crosslinking to construct lactams and other cyclic
structures. Other
derivatives include C-terminal hydroxymethyl derivatives, o-modified
derivatives (e.g., C-terminal
hydroxymethyl benzyl ether), N-terminally modified derivatives including
substituted amides such
as alkylamides and hydrazides.
[00157] In some cases, one or more L-amino acids in an IL-10 polypeptide
is replaced with
one or more D-amino acids.
[00158] In some cases, an IL-10 polypeptide is a retroinverso analog (see,
e.g., Sela and
Zisman (1997) FASEB J. 11:449). Retro-inverso peptide analogs are isomers of
linear
polypeptides in which the direction of the amino acid sequence is reversed
(retro) and the chirality,
D- or L-, of one or more amino acids therein is inverted (inverso), e.g.,
using D-amino acids rather
than L-amino acids. (See, e.g., Jameson et al. (1994) Nature 368:744; and
Brady et al. (1994)
Nature 368:6920.
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[00159] An IL-10 polypeptide can include a "Protein Transduction Domain"
(PTD), which
refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic
molecule that
facilitates traversing a lipid bilayer, micelle, cell membrane, organelle
membrane, or vesicle
membrane. A PTD attached to another molecule facilitates the molecule
traversing a membrane,
for example going from extracellular space to intracellular space, or cytosol
to within an organelle.
In some embodiments, a PTD is covalently linked to the amino terminus of an IL-
10 polypeptide,
while in other embodiments, a PTD is covalently linked to the carboxyl
terminus of an IL-10
polypeptide. Exemplary protein transduction domains include, but are not
limited to, a minimal
undecapeptide protein transduction domain (corresponding to residues 47-57 of
HIV-1 TAT
comprising YGRKKRRQRRR; SEQ ID NO:3); a polyarginine sequence comprising a
number of
arginine residues sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7,
8, 9, 10, or 10-50
arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-
96); a Drosophila
Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes
52(7):1732-1737); a
truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research
21:1248-1256);
polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008);
RRQRRTSKLMKR (SEQ ID NO:4); Transportan GWTLNSAGYLLGKINLKALAALAKKIL
(SEQ ID NO:5); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:6); and
RQIKIWFQNRRMKWKK (SEQ ID NO:7). Exemplary PTDs include, but are not limited
to,
YGRKKRRQRRR (SEQ ID NO:3), RKKRRQRRR (SEQ ID NO:8); an arginine homopolymer of
from 3 arginine residues to 50 arginine residues; exemplary PTD domain amino
acid sequences
include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID
NO:3);
RKKRRQRR (SEQ ID NO:9); YARAAARQARA (SEQ ID NO:10); THRLPRRRRRR (SEQ ID
NO:11); and GGRRARRRRRR (SEQ ID NO:12).
[00160] The carboxyl group COR3 of the amino acid at the C-terminal end of
an IL-10
polypeptide can be present in a free form (R3 = OH) or in the form of a
physiologically-tolerated
alkaline or alkaline earth salt such as, e.g., a sodium, potassium or calcium
salt. The carboxyl
group can also be esterified with primary, secondary or tertiary alcohols such
as, e.g., methanol,
branched or unbranched Ci-C6-alkyl alcohols, e.g., ethyl alcohol or tert-
butanol. The carboxyl
group can also be amidated with primary or secondary amines such as ammonia,
branched or
unbranched Ci-C6-alkylamines or C1-C6 di-alkylamines, e.g., methylamine or
dimethylamine.
[00161] The amino group of the amino acid NR1R2 at the N-terminus of an IL-
10
polypeptide can be present in a free form (R1= H and R2 = H) or in the form of
a physiologically-
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tolerated salt such as, e.g., a chloride or acetate. The amino group can also
be acetylated with
acids such that R1 = H and R2 = acetyl, trifluoroacetyl, or adamantyl. The
amino group can be
present in a form protected by amino-protecting groups conventionally used in
peptide chemistry,
such as those provided above (e.g., Fmoc, Benzyloxy-carbonyl (Z), Boc, and
Alloc). The amino
group can be N-alkylated in which R1 and/or R2 = C1-C6 alkyl or C2-C8 alkenyl
or C7-C9 aralkyl.
Alkyl residues can be straight-chained, branched or cyclic (e.g., ethyl,
isopropyl and cyclohexyl,
respectively).
Pegylation of IL-10
[00162] Pegylation of IL-10 comprises conjugating or linking the IL-10
polypeptide
sequence to any of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol (PEG),
polypropylene glycol, or polyoxyalkylenes. This is frequently effected by a
linking moiety
covalently bound to both the protein and the nonproteinaceous polymer, e.g., a
PEG. Such PEG-
conjugated biomolecules have been shown to possess clinically useful
properties, including better
physical and thermal stability, protection against susceptibility to enzymatic
degradation,
increased solubility, longer in vivo circulating half-life and decreased
clearance, reduced
immunogenicity and antigenicity, and reduced toxicity. In addition to the
beneficial effects of
pegylation on pharmacokinetic parameters, pegylation itself can enhance
activity. For example,
PEG-IL-10 has been shown to be more efficacious against certain cancers than
unpegylated IL-10
(see, e.g., EP 206636A2).
[00163] PEGs suitable for conjugation to a polypeptide sequence are
generally soluble in
water at room temperature, and have the general formula R(O-CH2-CH2)õ0-R,
where R is
hydrogen or a protective group such as an alkyl or an alkanol group, and where
n is an integer
from 1 to 1000. When R is a protective group, it generally has from 1 to 8
carbons. The PEG
conjugated to the polypeptide sequence can be linear or branched. Branched PEG
derivatives,
"star-PEGs" and multi-armed PEGs are contemplated by the present disclosure. A
molecular
weight of the PEG used in the present disclosure is not restricted to any
particular range, and
examples are set forth elsewhere herein; by way of example, certain
embodiments have molecular
weights between 5kDa and 20kDa, while other embodiments have molecular weights
between
4kDa and 10kDa.
[00164] The present disclosure also contemplates compositions of
conjugates wherein the
PEGs have different n values, and thus the various different PEGs are present
in specific ratios.
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For example, some compositions comprise a mixture of conjugates where n=1, 2,
3 and 4. In
some compositions, the percentage of conjugates where n=1 is 18-25%, the
percentage of
conjugates where n=2 is 50-66%, the percentage of conjugates where n=3 is 12-
16%, and the
percentage of conjugates where n=4 is up to 5%. Such compositions can be
produced by reaction
conditions and purification methods know in the art. Exemplary reaction
conditions are described
throughout the specification. Cation exchange chromatography can be used to
separate
conjugates, and a fraction is then identified which contains the conjugate
having, for example, the
desired number of PEGs attached, purified free from unmodified protein
sequences and from
conjugates having other numbers of PEGs attached.
[00165] Pegylation most frequently occurs at the alpha amino group at the
N-terminus of
the polypeptide, the epsilon amino group on the side chain of lysine residues,
and the imidazole
group on the side chain of histidine residues. Since most recombinant
polypeptides possess a
single alpha and a number of epsilon amino and imidazole groups, numerous
positional isomers
can be generated depending on the linker chemistry. General pegylation
strategies known in the
art can be applied herein.
[00166] Two widely used first generation activated monomethoxy PEGs
(mPEGs) are
succinimdyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992)
Biotechnol. Appl. Biochem
15:100-114; and Miron and Wilcheck (1993) Bio-conjug. Chem. 4:568-569) and
benzotriazole
carbonate PEG (BTC-PEG; see, e.g., Dolence, et al. US Patent No. 5,650,234),
which react
preferentially with lysine residues to form a carbamate linkage, but are also
known to react with
histidine and tyrosine residues. The linkage to histidine residues on certain
molecules (e.g., IFN-
a) has been shown to be a hydrolytically unstable imidazolecarbamate linkage
(see, e.g., Lee and
McNemar, U.S. Patent No. 5,985,263). Second generation pegylation technology
has been
designed to avoid these unstable linkages as well as the lack of selectivity
in residue reactivity.
Use of a PEG-aldehyde linker targets a single site on the N-terminus of a
polypeptide through
reductive amination.
[00167] PEG can be bound to a polypeptide of the present disclosure via a
terminal reactive
group (a "spacer") which mediates a bond between the free amino or carboxyl
groups of one or
more of the polypeptide sequences and polyethylene glycol. The PEG having the
spacer which
can be bound to the free amino group includes N-hydroxysuccinylimide
polyethylene glycol,
which can be prepared by activating succinic acid ester of polyethylene glycol
with N-
hydroxysuccinylimide. Another activated polyethylene glycol which can be bound
to a free amino
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group is 2,4-bis(0-methoxypolyethyleneglycol)-6-chloro-s-triazine, which can
be prepared by
reacting polyethylene glycol monomethyl ether with cyanuric chloride. The
activated
polyethylene glycol which is bound to the free carboxyl group includes
polyoxyethylenediamine.
[00168] Conjugation of one or more of the polypeptide sequences of the
present disclosure
to PEG having a spacer can be carried out by various conventional methods. For
example, the
conjugation reaction can be carried out in solution at a pH of from 5 to 10,
at temperature from
4 C to room temperature, for 30 minutes to 20 hours, utilizing a molar ratio
of reagent to protein
of from 4:1 to 30:1. Reaction conditions can be selected to direct the
reaction towards producing
predominantly a desired degree of substitution. In general, low temperature,
low pH (e.g., pH=5),
and short reaction time tend to decrease the number of PEGs attached, whereas
high temperature,
neutral to high pH (e.g., pH>7), and longer reaction time tend to increase the
number of PEGs
attached. Various means known in the art can be used to terminate the
reaction. In some
embodiments the reaction is terminated by acidifying the reaction mixture and
freezing at, e.g., -
20 C. Pegylation of various molecules is discussed in, for example, U.S. Pat.
Nos. 5,252,714;
5,643,575; 5,919,455; 5,932,462; and 5,985,263. PEG-IL-10 is described in,
e.g., U.S. Pat. No.
7,052,686. Specific reaction conditions contemplated for use herein are set
forth in the
Experimental section.
[00169] The present disclosure also contemplates the use of PEG mimetics.
Recombinant
PEG mimetics have been developed that retain the attributes of PEG (e.g.,
enhanced serum half-
life) while conferring several additional advantageous properties. By way of
example, simple
polypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser and Thr)
capable of forming
an extended conformation similar to PEG can be produced recombinantly already
fused to the
peptide or protein drug of interest (e.g., Amunix' XTEN technology; Mountain
View, CA). This
obviates the need for an additional conjugation step during the manufacturing
process. Moreover,
established molecular biology techniques enable control of the side chain
composition of the
polypeptide chains, allowing optimization of immunogenicity and manufacturing
properties.
[00170] Linkers: Linkers and their use have been described above. Suitable
linkers include
"flexible linkers" which are generally of sufficient length to permit some
movement between the
modified polypeptide sequences and the linked components and molecules. The
linker molecules
are generally about 6-50 atoms long. The linker molecules may also be, for
example, aryl
acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines,
diacids, amino
acids, or combinations thereof Suitable linkers can be readily selected and
can be of any suitable
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length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20,
20-30, 30-50 or more than
50 amino acids.
[00171] Examples of flexible linkers include glycine polymers (G),,
glycine-alanine
polymers, alanine-serine polymers, glycine-serine polymers (for example,
(Gõ,S0)õ, (GSGGS)õ
(SEQ ID NO:13), (Gõ,S0Gm),, (GmS0GmS0Gm),, (SEQ ID NO:14), (GSGGSm),, (SEQ ID
NO:15),
(GSGSmG)õ (SEQ ID NO:16) and (GGGSm)õ (SEQ ID NO:17), and combinations
thereof, where
m, n, and o are each independently selected from an integer of at least 1 to
20, e.g., 1-18, 2-16, 3-
14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8,9, or 10), and other flexible linkers.
Glycine and glycine-serine
polymers are relatively unstructured, and therefore may serve as a neutral
tether between
components. Examples of flexible linkers include, but are not limited to GGSG
(SEQ ID NO:18),
GGSGG (SEQ ID NO:19), GSGSG (SEQ ID NO:14), GSGGG (SEQ ID NO:20), GGGSG (SEQ
ID NO:21), and GSSSG (SEQ ID NO:22).
[00172] Additional examples of flexible linkers include glycine
polymers (G)n or
glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO:13), (GGGS)n (SEQ ID
NO:23)
and (GGGGS)n (SEQ ID NO:24), where n=1 to 50, for example, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 10-20,
20-30, 30-50). Exemplary flexible linkers include, but are not limited to GGGS
(SEQ ID NO:23),
GGGGS (SEQ ID NO:24), GGSG (SEQ ID NO:18), GGSGG (SEQ ID NO:19), GSGSG (SEQ ID
NO:14), GSGGG (SEQ ID NO:20), GGGSG (SEQ ID NO:21), and GSSSG (SEQ ID NO:22).
A
multimer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of
these linker sequences may be
linked together to provide flexible linkers that may be used to conjugate a
heterologous amino acid
sequence to the polypeptides disclosed herein.
Therapeutic and Prophylactic Uses
[00173] In particular embodiments, the present disclosure contemplates the
use of a PEG-
IL-10 and an IL-12 agent in the treatment and/or prevention of cancer-related
diseases, disorders
or conditions. While particular uses are described in detail hereafter, it is
to be understood that the
present disclosure is not so limited.
[00174] Representative cancers that may be treated or prevented using the
combination
therapies disclosed herein include cancer of the uterus, cervix, ovaries,
breast, prostate, testes,
gastrointestinal tract (e.g., esophagus, oropharynx, stomach, small or large
intestines, colon, or
rectum), kidney, renal cell, bladder, bone, bone marrow, skin, head or neck,
liver, gall bladder,
heart, lung, pancreas, salivary gland, adrenal gland, thyroid, brain (e.g.,
gliomas), ganglia, central
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nervous system (CNS) and peripheral nervous system (PNS), and cancers of the
immune system
(e.g., spleen or thymus).
[00175] The present disclosure also provides methods of treating or
preventing other
cancer-related diseases, disorders or conditions, including, for example,
immunogenic tumors,
non-immunogenic tumors, dormant tumors, virus-induced cancers (e.g.,
epithelial cell cancers,
endothelial cell cancers, squamous cell carcinomas and papillomavirus),
adenocarcinomas,
lymphomas (e.g., a B-cell lymphoma), leukemias, carcinomas, melanomas,
myelomas, sarcomas,
teratocarcinomas, chemically-induced cancers, and metastasis. In particular
embodiments, the
tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung
cancer, or
glioblastoma.
[00176] In further particular embodiments, the cancer is mammary
adenocarcinoma, lung
alveolar carcinoma, fibrosarcoma, and pulmonary metastasis of melanoma (Pegram
et al. (2012)
Advancements in Tumor Immunotherapy and Cancer Vaccines, Dr. Hilal Arnouk
(Ed.), ISBN:
978-953-307-998-1, InTech). Clinical studies exploring the antitumor effects
of IL-12 based
treatment in combination therapies or gene therapy include treatment of the
following tumors:
breast, pancreatic, hepatic, renal, cervical, gastrointestinal carcinomas,
colorectal, Non-Hodgkin's
lymphoma, melanoma (e.g., multiple melanoma), and AIDS-associated Kaposi
sarcoma (Lasek et
al. (2014) Cancer Immunol Immunother 63:419-35).
[00177] In particular embodiments of the present disclosure, the cancer-
related disease,
disorder or condition is an immune-insensitive tumor. Tumors that are
insensitive to therapeutic
immune manipulation may be described as exhibiting the following two
characteristics: 1) active
suppression of the immune system, and 2) an inflammatory response accompanied
by the
concomitant activation of immune-suppressive mechanisms resulting from
treatment thereof
(Galon et al. (July 25 2013) Immunity 39:11-26 (PubMed PMID: 238900600).
Examples of
immune-insensitive tumors include, but are not limited to, colon,
gastroesophageal, pancreatic and
breast cancer.
[00178] As described elsewhere herein, in some embodiments the present
disclosure
provides methods for treating a cancer-related disease, disorder or condition
with a PEG-IL-10 and
an IL-12 agent in combination with at least one additional therapeutic or
diagnostic agent,
examples of which are provided herein.
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Pharmaceutical Compositions
[00179] The PEG-IL-10 and IL-12 agents contemplated by the present
disclosure can be in
the form of compositions suitable for administration to a subject. In general,
such compositions
are "pharmaceutical compositions" comprising PEG-IL-10 and/or an IL-12 agent
and one or more
pharmaceutically acceptable or physiologically acceptable diluents, carriers
or excipients. In
certain embodiments, the PEG-IL-10 and IL-12 agents are each present in a
therapeutically
acceptable amount. The pharmaceutical compositions can be used in the methods
of the present
disclosure; thus, for example, the pharmaceutical compositions can be
administered ex vivo or in
vivo to a subject in order to practice the therapeutic and prophylactic
methods and uses described
herein.
[00180] In the description of the pharmaceutical compositions, and aspects
thereof, that
follows, the pharmaceutical compositions are generally described in the
context of a PEG-IL-10.
However, it is to be understood that the description also applies to the IL-12
agents of the present
disclosure, either in pharmaceutical compositions comprising combinations of a
PEG-IL-10 and
an IL-12 agent, or in pharmaceutical compositions comprising only one of the
components.
[00181] The pharmaceutical compositions of the present disclosure can be
formulated to be
compatible with the intended method or route of administration; exemplary
routes of
administration are set forth herein. Furthermore, the pharmaceutical
compositions can be used in
combination with other therapeutically active agents or compounds as described
herein in order to
treat or prevent the diseases, disorders and conditions as contemplated by the
present disclosure.
[00182] The pharmaceutical compositions typically comprise a
therapeutically effective
amount of a PEG-IL-10 and/or an IL-12 agent contemplated by the present
disclosure and one or
more pharmaceutically and physiologically acceptable formulation agents.
Suitable
pharmaceutically acceptable or physiologically acceptable diluents, carriers
or excipients include,
but are not limited to, antioxidants (e.g., ascorbic acid and sodium
bisulfate), preservatives (e.g.,
benzyl alcohol, methyl parabens, ethyl or n-propyl, p-hydroxybenzoate),
emulsifying agents,
suspending agents, dispersing agents, solvents, fillers, bulking agents,
detergents, buffers,
vehicles, diluents, and/or adjuvants. For example, a suitable vehicle can be
physiological saline
solution or citrate buffered saline, possibly supplemented with other
materials common in
pharmaceutical compositions for parenteral administration. Neutral buffered
saline or saline
mixed with serum albumin are further exemplary vehicles. Those skilled in the
art will readily
recognize a variety of buffers that can be used in the pharmaceutical
compositions and dosage
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forms contemplated herein. Typical buffers include, but are not limited to,
pharmaceutically
acceptable weak acids, weak bases, or mixtures thereof As an example, the
buffer components
can be water soluble materials such as phosphoric acid, tartaric acids, lactic
acid, succinic acid,
citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and
salts thereof. Acceptable
buffering agents include, for example, a Tris buffer, N-(2-
Hydroxyethyl)piperazine-N'-(2-
ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-
Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-
Morpholino)propanesulfonic acid
(MOPS), and N-tris[Hydroxymethyl]methy1-3-aminopropanesulfonic acid (TAPS).
[00183] After a pharmaceutical composition has been formulated, it can be
stored in sterile
vials as a solution, suspension, gel, emulsion, solid, or dehydrated or
lyophilized powder. Such
formulations can be stored either in a ready-to-use form, a lyophilized form
requiring
reconstitution prior to use, a liquid form requiring dilution prior to use, or
other acceptable form.
In some embodiments, the pharmaceutical composition is provided in a single-
use container (e.g.,
a single-use vial, ampoule, syringe, or autoinjector (similar to, e.g., an
EpiPeng)), whereas a
multi-use container (e.g., a multi-use vial) is provided in other embodiments.
Any drug delivery
apparatus can be used to deliver a PEG-IL-10 or an IL-12 agent, including
implants (e.g.,
implantable pumps) and catheter systems, slow injection pumps and devices, all
of which are well
known to the skilled artisan. Depot injections, which are generally
administered subcutaneously
or intramuscularly, can also be utilized to release the polypeptides disclosed
herein over a defined
period of time. Depot injections are usually either solid- or oil-based and
generally comprise at
least one of the formulation components set forth herein. One of ordinary
skill in the art is
familiar with possible formulations and uses of depot injections.
[00184] The pharmaceutical compositions can be in the form of a sterile
injectable aqueous
or oleagenous suspension. This suspension can be formulated according to the
known art using
those suitable dispersing or wetting agents and suspending agents mentioned
herein. The sterile
injectable preparation can also be a sterile injectable solution or suspension
in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-
butane diol.
Acceptable diluents, solvents and dispersion media that can be employed
include water, Ringer's
solution, isotonic sodium chloride solution, Cremophor ELTM (BASF, Parsippany,
NJ) or
phosphate buffered saline (PBS), ethanol, polyol (e.g., glycerol, propylene
glycol, and liquid
polyethylene glycol), and suitable mixtures thereof. In addition, sterile,
fixed oils are
conventionally employed as a solvent or suspending medium. For this purpose,
any bland fixed
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oil can be employed, including synthetic mono- or diglycerides. Moreover,
fatty acids such as
oleic acid, find use in the preparation of injectables. Prolonged absorption
of particular injectable
formulations can be achieved by including an agent that delays absorption
(e.g., aluminum
monostearate or gelatin).
[00185] The pharmaceutical compositions containing the active ingredient
can be in a form
suitable for oral use, for example, as tablets, capsules, troches, lozenges,
aqueous or oily
suspensions, dispersible powders or granules, emulsions, hard or soft
capsules, or syrups,
solutions, microbeads or elixirs. In particular embodiments, an active
ingredient of an agent co-
administered with a PEG-IL-10 and/or an IL-12 agent described herein is in a
form suitable for
oral use. Pharmaceutical compositions intended for oral use can be prepared
according to any
method known to the art for the manufacture of pharmaceutical compositions,
and such
compositions can contain one or more agents such as, for example, sweetening
agents, flavoring
agents, coloring agents and preserving agents in order to provide
pharmaceutically elegant and
palatable preparations. Tablets, capsules and the like contain the active
ingredient in admixture
with non-toxic pharmaceutically acceptable excipients which are suitable for
the manufacture of
tablets. These excipients can be, for example, diluents, such as calcium
carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate; granulating and
disintegrating agents,
for example, corn starch, or alginic acid; binding agents, for example starch,
gelatin or acacia, and
lubricating agents, for example magnesium stearate, stearic acid or talc.
[00186] The tablets, capsules and the like suitable for oral
administration can be uncoated or
coated by known techniques to delay disintegration and absorption in the
gastrointestinal tract and
thereby provide a sustained action. For example, a time-delay material such as
glyceryl
monostearate or glyceryl distearate can be employed. They can also be coated
by techniques
known in the art to form osmotic therapeutic tablets for controlled release.
Additional agents
include biodegradable or biocompatible particles or a polymeric substance such
as polyesters,
polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides, polyglycolic
acid, ethylene-
vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate,
orlactide/glycolide
copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate
copolymers in order to
control delivery of an administered composition. For example, the oral agent
can be entrapped in
microcapsules prepared by coacervation techniques or by interfacial
polymerization, by the use of
hydroxymethylcellulose or gelatin-microcapsules or poly (methylmethacrolate)
microcapsules,
respectively, or in a colloid drug delivery system. Colloidal dispersion
systems include
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macromolecule complexes, nano-capsules, microspheres, microbeads, and lipid-
based systems,
including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
Methods for the
preparation of the above-mentioned formulations will be apparent to those
skilled in the art.
[00187] Formulations for oral use can also be presented as hard gelatin
capsules wherein the
active ingredient is mixed with an inert solid diluent, for example, calcium
carbonate, calcium
phosphate, kaolin or microcrystalline cellulose, or as soft gelatin capsules
wherein the active
ingredient is mixed with water or an oil medium, for example peanut oil,
liquid paraffin, or olive
oil.
[00188] Aqueous suspensions contain the active materials in admixture with
excipients
suitable for the manufacture thereof. Such excipients can be suspending
agents, for example
sodium carboxymethylcellulose, methyl cellulose, hydroxy-
propylmethylcellulose, sodium
alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents, for
example a naturally-occurring phosphatide (e.g., lecithin), or condensation
products of an alkylene
oxide with fatty acids (e.g., polyoxy-ethylene stearate), or condensation
products of ethylene oxide
with long chain aliphatic alcohols (e.g., for heptadecaethyleneoxycetanol), or
condensation
products of ethylene oxide with partial esters derived from fatty acids and a
hexitol (e.g.,
polyoxyethylene sorbitol monooleate), or condensation products of ethylene
oxide with partial
esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene
sorbitan monooleate).
The aqueous suspensions can also contain one or more preservatives.
[00189] Oily suspensions can be formulated by suspending the active
ingredient in a
vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil,
or in a mineral oil such
as liquid paraffin. The oily suspensions can contain a thickening agent, for
example beeswax,
hard paraffin or cetyl alcohol. Sweetening agents such as those set forth
above, and flavoring
agents can be added to provide a palatable oral preparation.
[00190] Dispersible powders and granules suitable for preparation of an
aqueous suspension
by the addition of water provide the active ingredient in admixture with a
dispersing or wetting
agent, suspending agent and one or more preservatives. Suitable dispersing or
wetting agents and
suspending agents are exemplified herein.
[00191] The pharmaceutical compositions of the present disclosure can also
be in the form
of oil-in-water emulsions. The oily phase can be a vegetable oil, for example
olive oil or arachis
oil, or a mineral oil, for example, liquid paraffin, or mixtures of these.
Suitable emulsifying agents
can be naturally occurring gums, for example, gum acacia or gum tragacanth;
naturally occurring
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phosphatides, for example, soy bean, lecithin, and esters or partial esters
derived from fatty acids;
hexitol anhydrides, for example, sorbitan monooleate; and condensation
products of partial esters
with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.
[00192] Formulations can also include carriers to protect the composition
against rapid
degradation or elimination from the body, such as a controlled release
formulation, including
implants, liposomes, hydrogels, prodrugs and microencapsulated delivery
systems. For example,
a time delay material such as glyceryl monostearate or glyceryl stearate
alone, or in combination
with a wax, can be employed.
[00193] The present disclosure contemplates the administration of the IL-
10 polypeptides in
the form of suppositories for rectal administration. The suppositories can be
prepared by mixing
the drug with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid
at the rectal temperature and will therefore melt in the rectum to release the
drug. Such materials
include, but are not limited to, cocoa butter and polyethylene glycols.
[00194] The PEG-IL-10 and IL-12 agents contemplated by the present
disclosure can be in
the form of any other suitable pharmaceutical composition (e.g., sprays for
nasal or inhalation use)
currently known or developed in the future.
[00195] The concentration of a polypeptide or fragment thereof in a
formulation can vary
widely (e.g., from less than about 0.1%, usually at or at least about 2% to as
much as 20% to 50%
or more by weight) and will usually be selected primarily based on fluid
volumes, viscosities, and
subject-based factors in accordance with, for example, the particular mode of
administration
selected.
Routes of Administration
[00196] The present disclosure contemplates the administration of the PEG-
IL-10 and IL-12
agents, and compositions thereof, in any appropriate manner. Suitable routes
of administration
include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g.,
injection or implant),
intraperitoneal, intraci sternal, intraarticular, intraperitoneal,
intracerebral (intraparenchymal) and
intracerebroventricular), oral, nasal, vaginal, sublingual, intraocular,
rectal, topical (e.g.,
transdermal), sublingual and inhalation. Depot injections, which are generally
administered
subcutaneously or intramuscularly, can also be utilized to release the
polypeptides disclosed herein
over a defined period of time.
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[00197] Particular embodiments of the present disclosure contemplate
parenteral
administration. The parenteral administration is intravenous in some
embodiments and is
subcutaneous in others.
Supplementary Combination Therapy
[00198] The present disclosure contemplates the use of the combinations of
PEG-IL-10 and
an IL-12 agent in further combination with one or more active therapeutic
agents or other
prophylactic or therapeutic modalities (e.g., radiation). For purposes of this
application, such
further combinations are sometimes referred to as "supplementary
combinations", "supplementary
combination therapy", "combinations with an additional prophylactic or
therapeutic agent" and the
like, and agents that are added to combinations of PEG-IL-10 and an IL-12
agent can be referred
to as "supplementary agents" and the like. In such supplementary combination
therapy, the
various supplementary active agent(s) frequently have different mechanisms of
action than a PEG-
IL-10 and/or an IL-12 agent. Such supplementary combination therapy can be
especially
advantageous by allowing a dose reduction of one or more of the agents,
thereby reducing or
eliminating the adverse effects associated with one or more of the agents;
furthermore, such
supplementary combination therapy can have a synergistic therapeutic or
prophylactic effect on
the underlying proliferative disease, disorder, or condition. In some
embodiments of the present
disclosure the supplementary agent(s) is a diagnostic agent(s).
[00199] In particular embodiments, the present disclosure provides methods
for treating
and/or preventing cancer-related diseases, disorders or conditions with a PEG-
IL-10 and an IL-12
agent, and at least one additional therapeutic or diagnostic agent.
[00200] In some embodiments of the present disclosure, each of the PEG-IL-
10, the IL-12
agent and the supplementary agent(s) can be in a separate dosage form. By way
of example, the
PEG-IL-10 can be in a formulation suitable for SC administration, the IL-12
agent can be in a
formulation suitable for IV administration, and the supplementary agent can be
in a formulation
suitable for oral administration; in this context, each of the agents can be
housed separately or two
or more of the agents can be housed together (e.g., as distinct components of
a kit). In other
embodiments of the present disclosure, two or more of the PEG-IL-10, the IL-12
agent and the
supplementary agent(s) are in the same dosage form. For example, the PEG-IL-
10, the IL-12
agent, and the supplementary agent(s) can be formulated for IV administration;
in this context, one
or more of the agents can be co-formulated (e.g., as the active therapeutic
agents in a syringe).
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[00201] In certain embodiments, the PEG-IL-10, the IL-12 agent, and the
supplemental
agent(s) (e.g., a chemotherapeutic agent) are administered or applied
sequentially, e.g., where the
PEG-IL-10 is administered first, an IL-12 agent is administered second, and a
supplemental agent
is administered last. In other embodiments, the PEG-IL-10, the IL-12 agent,
and the supplemental
agent(s) are administered simultaneously, e.g., where two of them are
administered simultaneously
and the third is administered either before or after. Regardless of whether
the PEG-IL-10, the IL-
12 agent, and the supplemental agent(s) are administered sequentially,
simultaneously, or some
variation thereof, they are considered to be administered as supplementary
combination therapy
for purposes of the present disclosure.
[00202] The present disclosure contemplates the use of any possible dosing
regimen for the
supplementary combination therapy that may be acceptable, appropriate or
optimal under the
circumstances. The regimens described hereafter are exemplary, not
exclusionary. In one
embodiment, treatment with the PEG-IL-10, an IL-12 agent, and the supplemental
agent(s) are
maintained over a period of time. In another embodiment, treatment with the
PEG-IL-10, an IL-
12 agent, and the supplemental agent(s) are reduced or continued over a period
to time (e.g., when
the subject is stable). In another embodiment, treatment with the supplemental
agent(s) is reduced
or discontinued (e.g., when the subject is stable), while treatment with the
PEG-IL-10 and an IL-
12 agent is maintained at a constant dosing regimen. In a further embodiment,
treatment with the
supplemental agent(s) is reduced or discontinued (e.g., when the subject is
stable), treatment with
the PEG-IL-10 is reduced (e.g., lower dose, less frequent dosing or shorter
treatment regimen),
and treatment with the IL-12 agent is maintained at a constant dosing regimen.
In a further
embodiment, treatment with the supplemental agent(s) is reduced or
discontinued (e.g., when the
subject is stable), treatment with the PEG-IL-10 is reduced (e.g., lower dose,
less frequent dosing
or shorter treatment regimen), and treatment with IL-12 agent is maintained at
a constant dosing
regimen.
[00203] In yet another embodiment, treatment with the supplemental
agent(s) and the PEG-
IL-10 is maintained at a constant dosing regimen, while treatment with the IL-
12 agent is reduced
or discontinued (e.g., when the subject is stable). In yet a further
embodiment, treatment with the
supplemental agent(s) and the IL-12 agent is maintained at a constant dosing
regimen, while
treatment with the PEG-IL-10 is reduced or discontinued (e.g., lower dose,
less frequent dosing or
shorter treatment regimen). Identification and use of other dosing regimens
will be apparent to the
skilled artisan.
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[00204] While particular agents suitable for use with the combinations of
a PEG-IL-10 and
an IL-12 agent disclosed herein are set forth hereafter, it is to be
understood that the present
disclosure is not so limited. By way of example, but not limitation, a
prophylactic or therapeutic
agent may be a chemotherapeutic agent, an immune- or inflammation-related
agent, a metabolic
agent, an antiviral agent or an anti-thrombotic agent. The methods of the
present disclosure may
also be used in combination with non-pharmacological agents (e.g., radiology).
[00205] In a particular embodiment, the present disclosure contemplates
the use of a PEG-
IL-10 and an IL-12 agent with a chemotherapeutic agent(s) for treating and/or
preventing cancer,
tumor, or precancerous or cancer-associated disease, disorder or condition.
Examples of
chemotherapeutic agents include, but are not limited to, alkylating agents
such as thiotepa and
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such
as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines
including altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such
as
chiorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine,
prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine,
lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins,
actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,
carzinophilin,
chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-
norleucine,
doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,
mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,
rodorubicin,
streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-
metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as
denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine, 6-
mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;
androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such
as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;
bestrabucil; bisantrene;
edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone;
mopidamol;
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nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-
ethylhydrazide; procarbazine;
razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-
trichlorotriethylamine;
urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman;
gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g.,
paclitaxel and
doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum and
platinum coordination complexes such as cisplatin and carboplatin;
vinblastine; etoposide (VP-
16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;
navelbine; novantrone;
teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase
inhibitors;
difluoromethylornithine (DMF0); retinoic acid; esperamicins; capecitabine; and
pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[00206] Chemotherapeutic agents also include anti-hormonal agents that act
to regulate or
inhibit hormone action on tumors such as anti-estrogens, including for example
tamoxifen,
raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,
trioxifene, keoxifene,
onapristone, and toremifene; and antiandrogens such as flutamide, nilutamide,
bicalutamide,
leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or
derivatives of any of the
above. In certain embodiments, combination therapy comprises administration of
a hormone or
related hormonal agent.
[00207] Any other agent useful in the treatment or prevention of the
cancerous conditions
described herein is contemplated as a supplementary agent, including, but not
limited to, a
cytokine or cytokine antagonist, such as IL-12, INFa, or anti-epidermal growth
factor receptor,
radiotherapy, a monoclonal antibody against another tumor antigen, a complex
of a monoclonal
antibody and toxin, a T-cell adjuvant, bone marrow transplant, or antigen
presenting cells (e.g.,
dendritic cell therapy). Vaccines (e.g., as a soluble protein or as a nucleic
acid encoding the
protein) are also provided herein.
[00208] In particular embodiments, the additional prophylactic or
therapeutic agent is a
chemotherapeutic agent, examples of which are set forth herein. In some
embodiments, the
chemotherapeutic agent is a platinum-based antineoplastic, also referred to as
a platinum
coordination complex. These platinum-based antineoplastic agents crosslink
DNA, thereby
inhibiting DNA repair and/or DNA synthesis in cancer cells. Examples of such
agents include
cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin and
triplatin
[00209] The present disclosure encompasses pharmaceutically acceptable
salts, acids or
derivatives of any of the above.
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Dosing
[00210] A PEG-IL-10 and an IL-12 agent of the present disclosure can be
administered to a
subject in an amount that is dependent upon, for example, the goal of the
administration (e.g., the
degree of resolution desired); the age, weight, sex, and health and physical
condition of the subject
the formulation being administered; the route of administration; and the
nature of the disease,
disorder, condition or symptom thereof The dosing regimen can also take into
consideration the
existence, nature, and extent of any adverse effects associated with the
agent(s) being
administered. Effective dosage amounts and dosage regimens can readily be
determined from, for
example, safety and dose-escalation trials, in vivo studies (e.g., animal
models), and other methods
known to the skilled artisan.
[00211] As discussed elsewhere herein, the present disclosure contemplates
embodiments
of the PEG-IL-10 and IL-12 agent combination therapy wherein a PEG-IL-10 is
administered in
an amount and frequency such that a desired serum trough concentration (e.g.,
> 10 ng/mL) is
maintained. Embodiments of the of the PEG-IL-10 and IL-12 agent combination
therapy are also
contemplated wherein an IL-12 agent is dosed such that the serum concentration
achieves a peak
and is then cleared to an unmeasurable level before it is administered again.
[00212] In general, dosing parameters dictate that the dosage amount be
less than an
amount that could be irreversibly toxic to the subject (i.e., the maximum
tolerated dose, "MTD")
and not less than an amount required to produce a measurable effect on the
subject. Such amounts
are determined by, for example, the pharmacokinetic and pharmacodynamic
parameters associated
with ADME, taking into consideration the route of administration and other
factors.
[00213] As used herein, the term "EC50" and the phrase "half maximal
effective
concentration" have their generally accepted meaning; that is, the EC50 is the
concentration of a
therapeutic agent (e.g., a PEG-IL-10) which induces a response halfway between
the baseline and
the maximum after some specified exposure time. The skilled artisan is
familiar with means for
determining the EC50 of a therapeutic agent. For example, the EC50 may be
determined using
commercially available software (e.g., Graphpad Software, Inc.; La Jolla, CA)
after measuring
certain concentration-related parameters of the therapeutic agent in a cell-
based assay.
[00214] An effective dose (ED) is the dose or amount of an agent that
produces a
therapeutic response or desired effect in some fraction of the subjects taking
it. The "median
effective dose" or ED50 of an agent is the dose or amount of an agent that
produces a therapeutic
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response or desired effect in 50% of the population to which it is
administered. Although the
ED50 is commonly used as a measure of reasonable expectance of an agent's
effect, it is not
necessarily the dose that a clinician might deem appropriate taking into
consideration all relevant
factors. Thus, in some situations the effective amount can be more than the
calculated ED50, in
other situations the effective amount can be less than the calculated ED50,
and in still other
situations the effective amount can be the same as the calculated EDS .
[00215] In addition, an effective dose of a PEG-IL-10 and an IL-12 agent
of the present
disclosure can be an amount that, when administered in one or more doses to a
subject, produces a
desired result relative to a healthy subject. For example, for a subject
experiencing a particular
disorder, an effective dose can be one that improves a diagnostic parameter,
measure, marker and
the like of that disorder by at least about 5%, at least about 10%, at least
about 20%, at least about
25%, at least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about
70%, at least about 80%, at least about 90%, or more than 90%, where 100% is
defined as the
diagnostic parameter, measure, marker and the like exhibited by a normal
subject.
[00216] The amount of a PEG-IL-10 and an IL-12 agent necessary to treat a
disease,
disorder or condition described herein can be determined by activity assays
known in the art. By
way of example, in the tumor context, suitable IL-10 activity includes, for
example, CD8+ T-cell
infiltrate into tumor sites, expression of inflammatory cytokines, such as IFN-
y, IL-4, IL-6, IL-10,
and RANK-L, from these infiltrating cells, and increased levels of TNFa or
IFNy in biological
samples.
[00217] The therapeutically effective amount of PEG-IL-10 can range from
about 0.01 to
about 100 tg protein/kg of body weight/day, from about 0.1 to 20 tg protein/kg
of body
weight/day, from about 0.5 to 10 tg protein/kg of body weight/day, or about 1
to 4 tg protein/kg
of body weight/day. The present disclosure contemplates embodiments wherein
the amount of the
PEG-IL-10 component of the combination therapy is from 10.0 l.g/kg/day to 20.0
l.g/kg/day. In
some embodiments, the amount of the PEG-IL-10 administered is from 12.0
pg/kg/day to 18.0
pig/kg/day.
[00218] In some embodiments, PEG-IL-10 component of the combination
therapy is
administered (e.g., by continuous infusion) so as to provide for delivery of
about 50 to 800 tg
protein/kg of body weight/day (e.g., about 1 to 16 tg protein/kg of body
weight/day of PEG-IL-
10). Where delivered by infusion, the infusion rate can be varied based on
evaluation of, for
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example, adverse effects and blood cell counts. Other specific dosing
parameters for a PEG-IL-10
are described elsewhere herein.
[00219] The present disclosure contemplates embodiments wherein the amount
of the IL-12
component of the PEG-IL-10 combination therapy that is administered to the
subject to treat or
prevent a cancer-related disease, disorder or condition is from 0.01 g/kg/day
to 10.0 [tg/kg/day.
In other embodiments, the amount of the IL-12 agent is from 0.1 [tg/kg/day to
10.0 g/kg/day, and
in still other embodiments the amount of the IL-12 agent is from 1.0
[tg/kg/day to 10.0 g/kg/day.
In still further embodiments, the amount of the IL-12 component of the
combination therapy that
is administered to the subject to treat or prevent a cancer-related disease,
disorder or condition is
from 0.1 g/kg/day to 15.0 g/kg/day. In other embodiments, the amount of the
IL-12 agent is
from 1.0 g/kg/day to 15.0 g/kg/day, and in still other embodiments the
amount of the IL-12
agent is from 10.0 [tg/kg/day to 15.0 [tg/kg/day.
[00220] The present disclosure contemplates embodiments of the PEG-IL-
10/IL-12 agent
combination therapy in which the amount of PEG-IL-10 administered is 10.0
g/kg/day to 20.0
[tg/kg/day; 11.0 [tg/kg/day to 19.0 g/kg/day; 12.0 [tg/kg/day to 18.0
g/kg/day; 13.0 [tg/kg/day
to 17.0 g/kg/day; 14.0 g/kg/day to 16.0 [tg/kg/day; or about 15.0
[tg/kg/day. The present
disclosure contemplates embodiments of the PEG-IL-10/IL-12 agent combination
therapy in
which the amount of IL-12 agent administered is 0.01 [tg/kg/day to 10.0
g/kg/day; from 0.05
[tg/kg/day to 9.5 g/kg/day; 0.1 [tg/kg/day to 10.0 [tg/kg/day; 0.1 [tg/kg/day
to 9.0 g/kg/day; 0.5
[tg/kg/day to 8.5 g/kg/day; 1.0 [tg/kg/day to 10.0 [tg/kg/day; 1.0 [tg/kg/day
to 8.0 g/kg/day; 1.5
[tg/kg/day to 7.5 g/kg/day; 2.0 [tg/kg/day to 7.0 [tg/kg/day; 2.5 [tg/kg/day
to 6.5 [tg/kg/day; 3.0
[tg/kg/day to 6.0 g/kg/day; 3.5 [tg/kg/day to 5.5 [tg/kg/day; 4.0 [tg/kg/day
to 5.0 [tg/kg/day; or
4.5 [tg/kg/day, which may be administered in combination with any of the
amounts of PEG-IL-10
set out herein (e.g., PEG-IL-10 administered in an amount of 10.0 g/kg/day to
20.0 [tg/kg/day;
11.0 [tg/kg/day to 19.0 g/kg/day; 12.0 g/kg/day to 18.0 g/kg/day; 13.0
g/kg/day to 17.0
[tg/kg/day; 14.0 [tg/kg/day to 16.0 [tg/kg/day; or about 15.0 [tg/kg/day).
[00221] For administration of an oral agent, the compositions can be
provided in the form
of tablets, capsules and the like containing from 1.0 to 1000 milligrams of
the active ingredient,
particularly 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0,
200.0, 250.0, 300.0,
400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active
ingredient.
[00222] In certain embodiments, the dosage of the disclosed PEG-IL-10
and/or IL-12 agent
is contained in a "unit dosage form". The phrase "unit dosage form" refers to
physically discrete
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units, each unit containing a predetermined amount of a PEG-IL-10 and/or an IL-
12 agent of the
present disclosure, either alone or in combination with one or more additional
agents, sufficient to
produce the desired effect. It will be appreciated that the parameters of a
unit dosage form will
depend on the particular agent and the effect to be achieved.
Kits
[00223] The present disclosure also contemplates kits comprising PEG-IL-10
and/or an IL-
12 agent, and pharmaceutical compositions thereof. The kits are generally in
the form of a
physical structure housing various components, as described below, and can be
utilized, for
example, in practicing the methods described above. One or more components of
a kit can be in a
sterile container (e.g., a sterile vial).
[00224] A kit can include a PEG-IL-10 and/or an IL-12 agent disclosed
herein, which can
be in the form of a pharmaceutical composition suitable for administration to
a subject. The PEG-
IL-10 and/or IL-12 agent can be provided in a form that is ready for use or in
a form requiring, for
example, reconstitution or dilution prior to administration. When the PEG-IL-
10 and/or IL-12
agent is in a form that needs to be reconstituted by a user, the kit can also
include buffers,
pharmaceutically acceptable excipients, and the like, packaged with or
separately from the PEG-
IL-10 and/or IL-12 agent. A kit can also contain both the PEG-IL-10 and an IL-
12 agent as
described herein; the kit can contain the several agents separately or they
can already be combined
in the kit. Similarly, when supplementary therapy (e.g., a PEG-IL-10, an IL-12
agent, and a
supplementary agent) is contemplated, the kit can contain the several agents
separately or two or
more of them can already be combined in the kit. A kit of the present
disclosure can be designed
for conditions necessary to properly maintain the components housed therein
(e.g., refrigeration or
freezing).
[00225] A kit can contain a label or packaging insert including
identifying information for
the components therein and instructions for their use (e.g., dosing
parameters, clinical
pharmacology of the active ingredient(s), including mechanism(s) of action,
pharmacokinetics and
pharmacodynamics, adverse effects, contraindications, etc.). Each component of
the kit can be
enclosed within an individual container, and all of the various containers can
be within a single
package. Labels or inserts can include manufacturer information such as lot
numbers and
expiration dates. The label or packaging insert can be, e.g., integrated into
the physical structure
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housing the components, contained separately within the physical structure, or
affixed to a
component of the kit (e.g., an ampule, syringe or vial).
[00226]
Labels or inserts can additionally include, or be incorporated into, a
computer
readable medium, such as a disk (e.g., hard disk, card, memory disk), optical
disk such as CD- or
DVD-ROM/RAM, DVD, MI33, magnetic tape, or an electrical storage media such as
RAM and
ROM or hybrids of these such as magnetic/optical storage media, FLASH media or
memory-type
cards. In some embodiments, the actual instructions are not present in the
kit, but means for
obtaining the instructions from a remote source, e.g., via an internet site,
are provided.
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EXPERIMENTAL
[00227] The following examples are put forth so as to provide those of
ordinary skill in the
art with a complete disclosure and description of how to make and use the
present invention, and
are not intended to limit the scope of what the inventors regard as their
invention nor are they
intended to represent that the experiments below were performed and are all of
the experiments
that can be performed. It is to be understood that exemplary descriptions
written in the present
tense were not necessarily performed, but rather that the descriptions can be
performed to generate
the data and the like described therein. Efforts have been made to ensure
accuracy with respect to
numbers used (e.g., amounts, temperature, etc.), but some experimental errors
and deviations
should be accounted for.
[00228] Unless indicated otherwise, parts are parts by weight, molecular
weight is weight
average molecular weight, temperature is in degrees Celsius ( C), and pressure
is at or near
atmospheric. Standard abbreviations are used, including the following: s or
sec = second(s); min
= minute(s); h or hr = hour(s); aa = amino acid(s); bp = base pair(s); kb =
kilobase(s); nt =
nucleotide(s); ng = nanogram; 1.tg = microgram; mg = milligram; g = gram; kg =
kilogram; dl or
dL = deciliter; pi or [IL = microliter; ml or mL = milliliter; 1 or L = liter;
nM = nanomolar; 11M =
micromolar; mM = millimolar; M = molar; kDa = kilodalton; i.m. =
intramuscular(ly); i.p. =
intraperitoneal(ly); SC or SQ = subcutaneous(ly); HPLC = high performance
liquid
chromatography; BW = body weight; U = unit; ns = not statistically
significant; PMA = Phorbol
12-myristate 13-acetate; PBS = phosphate-buffered saline; HSA = human serum
albumin; DMEM
= Dulbeco's Modification of Eagle's Medium; PBMCs = primary peripheral blood
mononuclear
cells; FBS = fetal bovine serum; FCS = fetal calf serum; HEPES = 4-(2-
hydroxyethyl)-1-
piperazineethanesulfonic acid; LPS = lipopolysaccharide; ATCC = American Type
Culture
Collection.
Materials and Methods.
[00229] The following general materials and methods were used, where
indicated, or can be
used in the Examples below:
[00230] Molecular Biology Procedures. Standard methods in molecular
biology are
described in the scientific literature (see, e.g., Sambrook and Russell (2001)
Molecular Cloning,
3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and
Ausubel, et al.
(2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,
Inc. New York,
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N.Y., which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1),
cloning in
mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression
(Vol. 3), and
bioinformatics (Vol. 4)).
[00231] Antibody-related Processes. Production, purification, and
fragmentation of
polyclonal and monoclonal antibodies are described (e.g., Harlow and Lane
(1999) Using
Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY);
standard techniques
for characterizing ligand/receptor interactions are available (see, e.g.,
Coligan et al. (2001) Current
Protocols in Immunology, Vol. 4, John Wiley, Inc., NY); methods for flow
cytometry, including
fluorescence-activated cell sorting (FACS), are available (see, e.g., Shapiro
(2003) Practical Flow
Cytometry, John Wiley and Sons, Hoboken, NJ); and fluorescent reagents
suitable for modifying
nucleic acids, including nucleic acid primers and probes, polypeptides, and
antibodies, for use,
e.g., as diagnostic reagents, are available (Molecular Probes (2003)
Catalogue, Molecular Probes,
Inc., Eugene, OR.; Sigma-Aldrich (2003) Catalogue, St. Louis, MO.). Further
discussion of
antibodies appears elsewhere herein.
[00232] Software. Software packages and databases for determining, e.g.,
antigenic
fragments, leader sequences, protein folding, functional domains,
glycosylation sites, and
sequence alignments, are available (see, e.g., GCG Wisconsin Package
(Accelrys, Inc., San Diego,
CA); and DeCypherTM (TimeLogic Corp., Crystal Bay, NV).
[00233] Pegylation. Pegylated IL-10 as described herein can be synthesized
by any means
known to the skilled artisan. Exemplary synthetic schemes for producing mono-
PEG-IL-10 and a
mix of mono-/di-PEG-IL-10 have been described (see, e.g., U.S. Patent No.
7,052,686; US Pat.
Publn. No. 2011/0250163; WO 2010/077853). Particular embodiments of the
present disclosure
comprise a mix of selectively pegylated mono- and di-PEG-IL-10. In addition to
leveraging her
own skills in the production and use of PEGs (and other drug delivery
technologies) suitable in the
practice of the present disclosure, the skilled artisan is familiar with many
commercial suppliers of
PEG-related technologies (e.g., NO America Corp (Irvine, CA) and Parchem (New
Rochelle,
NY)).
[00234] Mice. Various mice and other animal strains can be used in
conjunction with the
teachings of the present disclosure. For example, immunocompetent Balb/C or B-
cell ¨ deficient
Balb/C mice can be obtained from The Jackson Lab., Bar Harbor, ME and used in
accordance
with standard procedures (see, e.g., Martin et al (2001) Infect. Immun.,
69(11):7067-73 and
Compton et al. (2004) Comp. Med. 54(6):681-89). Other mice strains suitable
for the
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experimental work contemplated by the present disclosure are known to the
skilled artisan and are
generally available from The Jackson Lab or another supplier.
[00235] IL-10 Concentrations. Serum IL-10 concentration levels and
exposure levels can
be determined by standard methods used in the art. For example, a serum
exposure level assay
can be performed by collecting whole blood (-50 L/mouse) from mouse tail
snips into plain
capillary tubes, separating serum and blood cells by centrifugation, and
determining IL-10
exposure levels by standard ELISA kits and techniques.
[00236] The assays described hereafter are representative, and not
exclusionary.
[00237] In Vitro Cytokine Secretion Assay. Activated primary human CD8+ T-
cells
secrete IFN-y when treated with PEG-IL-10 and then with an anti-CD3 antibody.
The following
protocol provides an exemplary assay to examine cytokine secretion.
[00238] Human PBMCs can be isolated according to any standard protocol
(see, e.g., Fuss
et al. (2009) Current Protocols in Immunology, Unit 7.1, John Wiley, Inc.,
NY). CD8+ T-cells
can be isolated using Miltenyi Biotec's MACS cell separation technology
according to the
manufacture's protocol (Miltenyi Biotec; Auburn, CA). For assays during
activation, the isolated
CD8+ T-cells (2 x 106 cells/mL, 5 x 105 cells per well of a standard 96-well
plate) can be activated
with plate-bound anti-CD3 and anti-CD28 (plates are pre-coated with 10 .g/mL
anti-CD3 and 2
pg/mL anti-CD28; Affymetrix eBioscience; San Diego, CA) and appropriate
concentrations of IL-
12 or PEG-IL-10 for 3 days in AIM V media (Life Technologies; Carlsbad, CA).
The media can
then be collected and assayed for IFN-y using a commercially available ELISA
kit following the
manufacture's protocol (Affymetrix eBioscience; San Diego, CA). For assays
during the rest
phase, the isolated CD8+ T-cells (3 x 106 cells/mL, 3 x 106 cells per well of
a standard 24-well
plate) can be activated with plate-bound anti-CD3 and anti-CD28 (plates are
pre-coated with 10
.g/mL anti-CD3 and 2 pg/mL anti-CD28; Affymetrix eBioscience; San Diego, CA)
for 3 days.
Following activation, cells can then be collected, re-plated (2 x 106
cells/mL, 5 x 105 cells per well
of a standard 96-well plate) and treated with appropriate concentrations of IL-
12 or PEG-hIL-10
for 3 days in AIM V media. After treatment, cells can be collected, re-plated
(2 x 106 cells/mL, 5 x
105 cells per well of a standard 96-well plate) and treated with 1 .g/mL
soluble anti-CD3 for 4 hrs
in AIM V media. The media can then be collected and assayed for IFN-y
(Affymetrix
eBioscience; San Diego, CA), Granzyme B and Perforin (Mabtech; Cincinnati, OH)
using
commercially available ELISA kits following the manufacture's protocol.
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[00239] TNFa Inhibition Assay. PMA-stimulation of U937 cells (lymphoblast
human cell
line from lung available from Sigma-Aldrich (#85011440); St. Louis, MO) causes
the cells to
secrete TNFa, and subsequent treatment of these TNFa ¨ secreting cells with
human IL-10 causes
a decrease in TNFa secretion in a dose-dependent manner. An exemplary TNFa
inhibition assay
can be performed using the following protocol.
[00240] After culturing U937 cells in RMPI containing 10% FBS/FCS and
antibiotics, plate
1 x 105, 90% viable U937 cells in 96-well flat bottom plates (any plasma-
treated tissue culture
plates (e.g., Nunc; Thermo Scientific, USA) can be used) in triplicate per
condition. Plate cells to
provide for the following conditions (all in at least triplicate; for 'media
alone' the number of
wells is doubled because one-half will be used for viability after incubation
with 10 nM PMA): 5
ng/mL LPS alone; 5 ng/mL LPS + 0.1 ng/mL rhIL-10; 5 ng/mL LPS + 1 ng/mL rhIL-
10; 5 ng/mL
LPS + 10 ng/mL rhIL-10; 5 ng/mL LPS + 100 ng/mL rhIL-10; 5 ng/mL LPS + 1000
ng/mL rhIL-
10; 5 ng/mL LPS + 0.1ng/mL PEG-rhIL-10; 5 ng/mL LPS + 1 ng/mL PEG-rhIL-10; 5
ng/mL LPS
+ 10 ng/mL PEG-rhIL-10; 5 ng/mL LPS + 100 ng/mL PEG-rhIL-10; and 5 ng/mL LPS +
1000
ng/mL PEG-rhIL-10. Expose each well to 10 nM PMA in 200 !IL for 24 hours,
culturing at 37 C
in 5% CO2 incubator, after which time ¨90% of cells should be adherent. The
three extra wells
can be re-suspended, and the cells are counted to assess viability (>90%
should be viable). Wash
gently but thoroughly 3X with fresh, non-PMA ¨ containing media, ensuring that
cells are still in
the wells. Add 100 tL per well of media containing the appropriate
concentrations (2X as the
volume will be diluted by 100%) of rhIL-10 or PEG-rhIL-10, incubate at 37 C in
a 5% CO2
incubator for 30 minutes. Add 100 tL per well of 10 ng/mL stock LPS to achieve
a final
concentration of 5 ng/mL LPS in each well, and incubate at 37 C in a 5% CO2
incubator for 18-
24 hours. Remove supernatant and perform TNFa ELISA according to the
manufacturer's
instructions. Run each conditioned supernatant in duplicate in ELISA.
[00241] MC/9 Cell Proliferation Assay. IL-10 administration to MC/9 cells
(murine cell
line with characteristics of mast cells available from Cell Signaling
Technology; Danvers, MA)
causes increased cell proliferation in a dose-dependent manner. Thompson-
Snipes, L. et al. (1991)
J. Exp. Med. 173:507-10) describe a standard assay protocol in which MC/9
cells are
supplemented with IL3 + IL-10 and IL-3 + IL-4 + IL-10. Vendors (e.g., R&D
Systems, USA; and
Cell Signaling Technology, Danvers, MA) use the assay as a lot release assay
for rhIL-10. Those
of ordinary skill in the art will be able to modify the standard assay
protocol described in
Thompson-Snipes, L. et al, such that cells are only supplemented with IL-10.
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[00242] Tumor Models and Tumor Analysis. Any art-accepted tumor model,
assay, and the
like can be used to evaluate the effect of the IL-10 molecules described
herein on various tumors.
The tumor models and tumor analyses described hereafter are representative of
those that can be
utilized. Syngeneic mouse tumor cells are injected subcutaneously or
intradermally at 104, 105 or
106 cells per tumor inoculation. Ep2 mammary carcinoma, CT26 colon carcinoma,
PDV6
squamous carcinoma of the skin and 4T1 breast carcinoma models can be used
(see, e.g.,
Langowski et al. (2006) Nature 442:461-465). Immunocompetent Balb/C or B-cell
deficient
Balb/C mice can be used. PEG 10-mIL-10 can be administered to the
immunocompetent mice,
while PEG-hIL-10 treatment can be in the B-cell deficient mice. Tumors are
allowed to reach a
size of 100-250 mm3 before treatment is started. IL-10, PEG-mIL-10, PEG-hIL-
10, or buffer
control is administered SC at a site distant from the tumor implantation.
Tumor growth is
typically monitored twice weekly using electronic calipers. Tumor tissues and
lymphatic organs
are harvested at various endpoints to measure mRNA expression for a number of
inflammatory
markers and to perform immunohistochemistry for several inflammatory cell
markers. The tissues
are snap-frozen in liquid nitrogen and stored at -80 C. Primary tumor growth
is typically
monitored twice weekly using electronic calipers. Tumor volume can be
calculated using the
formula (width2 x length/2) where length is the longer dimension. Tumors are
allowed to reach a
size of 90-250 mm3 before treatment is started.
EXAMPLE 1
Anti-tumor Effect of PEG-IL-10 in Combination with IL-12
[00243] This example demonstrates the combinatorial effect of PEG-IL-10
and IL-12 on
tumor size in a murine 4T1 tumor model.
[00244] Briefly, 1 x 104 4T1 cells (CRL-2539; ATCC, Manassas, VA) in a
volume of 100
11.1 were implanted SC into the right lower flank of female BALB/c mice
(Jackson Laboratory, Bar
Harbor, ME) of 4-6 weeks of age. Once palpable, tumor growth was measured
twice weekly ¨
tumor volume can be calculated using the formula (width2 x length/2), where
length is the longer
dimension. When tumors reached an average of 75 mm3 in volume, animals were
stratified.
[00245] Eight mice per cohort were administered vehicle, and/or 1 mg/kg
PEG-rMuIL-10
(ARMO Biosciences, Redwood City, CA), and/or 0.05, 0.1, or 0.5 mg/kg rMuIL-12
(R&D
Systems, Minneapolis, MN) SC daily for 21 or 28 days. Each mouse received two
separate
injections (e.g., IL-10 and vehicle, or IL-10 and IL-12, or vehicle and
vehicle). After 21 days of
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dosing, 4 mice from each group were sacrificed for tissue and tumor analysis.
After 28 days of
dosing, the remaining mice from each group were sacrificed for tissue and
tumor analysis.
[00246] Tumor weights were assessed after 21 days, and the data are
presented in FIG. 2.
The amount of rMuIL-12 adminsitered is indicated on the X-axis; as noted above
where
PEG-rMuIL-10 was administered, the dose was 1 mg/kg. As indicated in FIG. 2,
administration of
each of the combinations of PEG-rMuIL-10 and rMuIL-12 resulted in a larger
reduction in tumor
weight than the administration of either agent alone. This effect was more
pronounced at the
higher doses of rMuIL-12 (i.e., 0.5 and 0.1 mg/kg; * = P <0.05). The bars in
FIG. 2 represent the
mean of the individual mouse data. Mice evaluated after 28 days exhibited the
same general
trends (data not shown).
EXAMPLE 2
Effect of PEG-IL-10 in Combination with IL-12 on Serum Cytokine Levels
[00247] This example demonstrates the combinatorial effect of PEG-IL-10
and IL-12 on
serum IFNy and TNFa levels in tumor-bearing mice. As described herein,
exposure to each of IL-
and IL-12 individually, and particularly IL-12, leads to the induction of the
serum cytokines
IFNy and TNFa. The increased serum levels of IFNy and TNFa (though primarily
IFNy) are
associated with IL-12's systemic toxicity.
[00248] Briefly, IFNy and TNFa levels were evaluated in the mice described
in Example 1
(i.e., mice administered vehicle, 1 mg/kg PEG-rMuIL-10, and/or 0.05, 0.1, or
0.5 mg/kg rMuIL-12
SC daily) after 9 days of dosing, 4 hours after dose administration. Plasma
cytokine levels were
detected using Meso Scale Discovery's V-PLEX Proinflammatory Panell (mouse)
Kit (Rockville,
Maryland), performed according to the manufacturer's instructions. The results
are provided in
FIG. 3A and FIG. 3B. The amount of rMuIL-12 adminsitered is indicated on the X-
axis in each of
FIGS. 3A and 3B; as noted above, where PEG-rMuIL-10 was administered, the dose
was 1 mg/kg.
[00249] As indicated in FIG. 3A, co-administration of PEG-rMuIL-10 with
each of the
three rMuIL-12 doses resulted in decreases in the serum IFNy levels observed
following
administration of each of the three rMuIL-12 doses alone. Moreover, when 0.5
mg/kg rMuIL-12
was co-administered with 1 mg/kg PEG-rMuIL-10, there was a statistically
signification decrease
(*** = P <0.001) in the serum IFNy levels as compared to administration of 0.5
mg/kg rMuIL-12
alone. These data are representative of the effect that PEG-IL-10 has when co-
administered with
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IL-12 ¨ the enhanced anti-tumor response resulting from combination therapy
(see FIG. 2) is not
compromised while the putative toxicity "index" associated with IL-12 is
reduced.
[00250] As indicated in FIG. 3B, co-administration of PEG-rMuIL-10 with
each of the
three rMuIL-12 doses resulted in decreases in the serum TNFa levels observed
following
administration of each of the three rMuIL-12 doses alone. Moreover, when 0.5
mg/kg rMuIL-12
was co-administered with 1 mg/kg PEG-rMuIL-10, there was a statistically
signification decrease
(*** = P <0.001) in the serum TNFa levels as compared to administration of 0.5
mg/kg rMuIL-12
alone. These data are representative of the effect that PEG-IL-10 has when co-
administered with
IL-12 ¨ the enhanced anti-tumor response resulting from combination therapy
(see FIG. 2) is not
compromised while the putative toxicity "index" associated with IL-12 is
reduced.
[00251] Particular embodiments of this invention are described herein,
including the best
mode known to the inventors for carrying out the invention. Upon reading the
foregoing,
description, variations of the disclosed embodiments may become apparent to
individuals working
in the art, and it is expected that those skilled artisans may employ such
variations as appropriate.
Accordingly, it is intended that the invention be practiced otherwise than as
specifically described
herein, and that the invention includes all modifications and equivalents of
the subject matter
recited in the claims appended hereto as permitted by applicable law.
Moreover, any combination
of the above-described elements in all possible variations thereof is
encompassed by the invention
unless otherwise indicated herein or otherwise clearly contradicted by
context.
[00252] All publications, patent applications, accession numbers, and
other references cited
in this specification are herein incorporated by reference as if each
individual publication or patent
application were specifically and individually indicated to be incorporated by
reference.
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